Cell-mediated immunity induced by rabies vaccination was studied in humans by the determination o... more Cell-mediated immunity induced by rabies vaccination was studied in humans by the determination of specific interleukin-2 (IL-2) production in a large number of donors (postexposure immunized patients and pre-exposure immunized laboratory workers). Peripheral blood lymphocytes (PBL) from 35 donors were tested for IL-2 production after in vitro stimulation by different rabies and rabies-related viruses. IL-2 responses were compared to antibody recognition of these different virus serotypes by sera from the same individuals. IL-2 was produced by PBL from more than 85% of donors after stimulation with inactivated and purified rabies viruses (IPR V)prepared from either Pittman Moore (PM)
The present report demonstrates that liposomes increase the interleukin-2 (IL-2) dependent prolif... more The present report demonstrates that liposomes increase the interleukin-2 (IL-2) dependent proliferation of cytotoxic T-lymphocyte line (CTLL) cells used for the measurement of IL-2 activity. This effect was better observed with suboptimal doses of IL-2 and low concentrations of lipids. The increased IL-2 dependent proliferation is not due to a direct effect of liposomes on CTLL cells but rather to an interaction between IL-2 and liposomes. An interaction between IL-2 and components of fetal calf serum is also demonstrated. The results indicate that liposomes may interfere with IL-2 bioassay but also show the possibility of potentiating IL-2 activity for therapeutic purposes.
The potency of different rabies vaccines was measured via cell mediated immunity (CMI) assessed b... more The potency of different rabies vaccines was measured via cell mediated immunity (CMI) assessed by the production of interleukin-2 (IL-2) by CD4+CD8-lymphocytes. IL-2 production by splenocytes from mice immunized with various vaccines was measured following in vitro stimulation with antigens from different rabies and rabies-related strains. IL-2 production was specific, reproducible and correlated with the vaccine protective activity as determined by the pre-exposure NIH test. Our results suggest that measurement of IL-2 production could be used for the appraisal of rabies vaccine potency.
Objetivo: Rever de forma sistemática as substâncias adjuvantes mais estudadas, utilizadas e as no... more Objetivo: Rever de forma sistemática as substâncias adjuvantes mais estudadas, utilizadas e as novas substâncias; com o intuito de atualizar os conceitos de sua ação por atuarem, juntamente com o antígeno, no sistema imunológico e seu futuro uso em vacinas humanas ou animais.
Rabies is an important viral disease with a great impact on public health due to its extensively ... more Rabies is an important viral disease with a great impact on public health due to its extensively fatal outcome. In endemic areas, rabies may be responsible for over 50,000 human fatalities per year, which is a high number, since a well-established prophylaxis by immunization is available. Despite experimental protocols, no antiviral drugs are currently effective against rabies virus infection. For this reason, antivirals directed to treat human rabies need to be developed. In order to standardize in vitro antiviral screening for rabies virus, we evaluated two methods: the inhibition of cytopathic effect (CPE) and the MTT assay. Anti-rabies activity was only measurable by CPE, while the MTT assay failed to detect antiviral activity in rabies infected McCoy cells. In addition, no significant correlation was observed when comparing both assays. The correlation between the inhibition of CPE and MTT assay was shown when Vero cells were infected with herpes simplex virus type 1 and Hep-2 cells were infected with adenovirus type 5, reinforcing that despite the reliable CPE, McCoy cells infected with rabies cannot be assessed by MTT. In conclusion, these results have shown that the inhibition of CPE should be used for anti-rabies screening in McCoy cells, while the MTT assay was unsuitable in this in vitro system..
Hantaviruses are rodent-borne RNA viruses that cause hemorrhagic fever with renal syndrome (HFRS)... more Hantaviruses are rodent-borne RNA viruses that cause hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). From the first detection of infection in Brazil in 1993 until 2009, 1161 cases of HPS have been reported, with mortality rates of around approximately 40%. Currently, due to the absence of a vaccine or specific antiviral therapy, the only way to reduce mortality by hantavirus infection is a fast and precise diagnosis that allows for supportive clinical health care. To improve the detection of hantavirus infection, we developed monoclonal antibodies (Mabs) against the nucleoprotein (rNDelta85) of the Araucaria hantavirus strain (ARAUV). The specificity of generated Mabs for rNDelta85 was demonstrated by western blot, indirect immunofluorescence and immunohistochemistry. These are the first monoclonal antibodies to be produced and characterized against the South American hantavirus strain, and may be of special interest in the development of diagnos...
Revista do Instituto de Medicina Tropical de São Paulo, 1999
The cellular and humoral immune responses of mice inoculated with rabies virus and treated with t... more The cellular and humoral immune responses of mice inoculated with rabies virus and treated with the Bacillus of Calmette-Guérin, Avridine and Propionibacterium acnes were evaluated in this paper. There was a higher percentage of surviving mice in groups submitted to P. acnes treatment. Lower levels of interferon-gamma (IFN-gamma) were found in infected mice. The intra-pad inoculation test (IPI) was not effective to detect cellular immune response, contrary to the results found in MIF reaction. The survival of mice did not present correlation with the levels of antirabies serum neutralizing (SN) antibodies titers, IFN-gamma concentration and MIF response.
Monoclonal antibodies (mAbs) can be used therapeutically by binding to molecular targets with hig... more Monoclonal antibodies (mAbs) can be used therapeutically by binding to molecular targets with high specificity. Therefore, they have excellent therapeutic applications in ophthalmology. This manuscript presents four aspects of the therapeutic use of mAbs in ophthalmology: the scientific rationale, the unique characteristics of selected mAbs, the current state-of-the-art application, and relevant therapeutic mAbs for future applications in ophthalmology. We identified in the literature various single-agent therapies that inhibit the following targets: tumor necrosis factor (TNF), epithelial growth factor receptor, vascular endothelial growth factor (VEGF) receptor, basic fibroblast growth factor receptor, platelet-derived growth factor, and cluster of differentiation antigens. The roles of all biochemical targets in ocular diseases were evaluated. Current and future mAbs against various cytokines were assessed for the treatment of ocular diseases. The medical literature showed the clinical benefits of mAbs for treating angiogenic and inflammatory ocular diseases. Two anti-VEGF mAbs, bevacizumab and ranibizumab, and three anti-TNF agents, infliximab, etanercept, and adalimumab, control ocular neovascularization and intraocular inflammation. Other mAbs such as rituximab, daclizumab, efalizumab, and alemtuzumab showed positive results in animal and early clinical studies and may represent useful adjuvant therapies for ocular lymphoma or ocular inflammation. Ranibizumab is the only FDA-approved therapy; for other mAbs the so-called off-label application remains the standard. Intravenous administration of mAbs has demonstrated acceptable toxicity profiles, while intraocular injection may decrease the chances of systemic complications and increase the amount of drug available to the retina and choroid. In conclusion, effective clinical use of mAbs in ophthalmology is more commonly seen in the field of angiogenic vitreoretinal and autoimmune inflammatory diseases. The challenge for the future is combining biologic therapies to improve the quality and duration of responses while diminishing side effects. The role of mAbs within ophthalmic treatments will be defined according to future clinical experience and the results of randomized clinical trials.
Using the laboratory mice, Fuenzalida-Palacios mouse brain human rabies vaccine was administered ... more Using the laboratory mice, Fuenzalida-Palacios mouse brain human rabies vaccine was administered in groups of animals previously inoculated with rabies virus and then submitted to treatments with the immunomodulators onco-BCG, avridine and Propionibacterium acnes. Humoral and cellular immune responses were evaluated through the macrophage inhibition factor (MIF), intra-pad inoculation (IPI) and serum neutralization (SN) tests and by the detection of g-interferon (IFN-g). The IPI test was not eective in detecting the response of delayed-type hypersensitivity, contrary to MIF, which showed the immune cellular response. Higher levels of IFN-g were observed in the groups of mice vaccinated and treated with avridine and P. acnes. Although immunomodulating activities have been detected, the use of adjuvants with the Fuenzalida-Palacios type vaccine in mice did not reveal any encouraging results. #
The glycoprotein (G) of rabies virus (RV) is important for virus infectivity and induction of the... more The glycoprotein (G) of rabies virus (RV) is important for virus infectivity and induction of the protective immunity. In this study, the region comprising linear epitopes (residues 179-281, ERA strain), named rGERA179-281, was cloned in frame with a hexahistidine tag coding sequence at its N-terminal end and overexpressed in Escherichia coli Rosetta strain. The expression under transcriptional regulation of T7 promoter yielded insoluble protein aggregates in the form of inclusion bodies. The inclusion bodies were solubilized with 6 M guanidine HCl and the protein was purified to homogeneity under denaturing conditions. Mass spectrometry data confirmed the identity of the protein. The purified protein (13.8 kDa) showed significant reactivity with antibodies present in a therapeutic human rabies immune globulin (HRIG), as demonstrated by immunoblotting analysis. In addition, by in vitro competitive neutralization assay, rGERA179-281 led to a measurable reduction in the ability of HRIG to neutralize rabies virus. These results, along with the good yield obtained, encourage further studies on the more detailed immunological properties of rGER-A179-281, such as the ability to induce rabies virus neutralizing antibodies and the production of anti-G monoclonal antibodies, which together, might be useful for the development of new diagnostic methods.
This work describes the cytopathic effect on cells, cytotoxic action on mice, and antiviral activ... more This work describes the cytopathic effect on cells, cytotoxic action on mice, and antiviral activity of cinnabarin. This substance had no effect on mouse neuroblastoma cells (NA cell, ATCC clone C-1300) at a concentration of 0.31 mg/ml, it was not able to cause toxic effects in mice at concentrations of 1000 mg/kg, and reduced by four times the titers of the rabies virus at concentrations of 0.31 mg/ml.
Ten monoclonal antibodies (MAbs) against rabies virus, including IgG3κ, IgG2aκ, IgMκ, and an IgG2... more Ten monoclonal antibodies (MAbs) against rabies virus, including IgG3κ, IgG2aκ, IgMκ, and an IgG2bκ isotype, were produced and characterized using neutralization, ELISA, immunodot-blot, and immunofluorescence assays. MAb 8D11, which recognized rabies virus glycoprotein, was found to neutralize rabies virus in vitro. When submitted to an immunofluorescence assay, seven MAbs showed different reactivity against 35 Brazilian rabies virus isolates. Three MAbs (LIA 02, 3E6, and 9C7) only failed to recognize one or two virus isolates, whereas MAb 6H8 was found to be reactive against all virus isolates tested. MAbs were also evaluated for their immunoreactivity against fixed rabies virus strains present in human and veterinary commercial vaccines. MAbs LIA 02, 6H8, and 9C7 reacted against all vaccine strains, while the remaining MAbs recognized at least 76% of vaccine strains tested. This research provides a set of MAbs with potential application for improving existing or developing new diagnostic tests and immunoassays.
Murine myeloma cell lines play an important role in different areas of scientific research and ar... more Murine myeloma cell lines play an important role in different areas of scientific research and are essential tools for monoclonal antibody production technology. Thus, it is important to understand the biology of these cell lines in order to provide useful information to various research fronts. The present study aims to perform detailed analyses of surface antigens expressed on three major murine myeloma cell lines extensively used for MAb production. The P3X63Ag8.653 cell line expresses molecules associated with T cell interaction (CD40(low), CD80(low)), as well as antigens related to plasma cell phenotype (CD138(high), CD184(low)). The Sp2/0-Ag14 cell line presents molecules associated with BCR activation and regulation (CD79b(low), CD22(low), CD72(med)), molecules related to T cell interaction (CD40(low), CD80(low)), and markers of plasma cell phenotype (CD138(high), CD184(low)). The NS1 cell line presents all molecules of plasma cell phenotype evaluated in this study (CD184(low), CD138(high), CD38(med)) with low expression of CD72 (CD72(low)), a molecule related to BCR activation. Molecules associated with immune response modulation such as CD23 and CD25, as well as CD117, a marker related to undifferentiated cell phenotype, were not observed in any of the three murine myeloma cell lines evaluated. These data show that in spite of their common origin and function, the immunological profiles differ between P3X63Ag8.653, Sp2/0-Ag14, and NS1 cell lines.
Cell-mediated immunity induced by rabies vaccination was studied in humans by the determination o... more Cell-mediated immunity induced by rabies vaccination was studied in humans by the determination of specific interleukin-2 (IL-2) production in a large number of donors (postexposure immunized patients and pre-exposure immunized laboratory workers). Peripheral blood lymphocytes (PBL) from 35 donors were tested for IL-2 production after in vitro stimulation by different rabies and rabies-related viruses. IL-2 responses were compared to antibody recognition of these different virus serotypes by sera from the same individuals. IL-2 was produced by PBL from more than 85% of donors after stimulation with inactivated and purified rabies viruses (IPR V)prepared from either Pittman Moore (PM)
The present report demonstrates that liposomes increase the interleukin-2 (IL-2) dependent prolif... more The present report demonstrates that liposomes increase the interleukin-2 (IL-2) dependent proliferation of cytotoxic T-lymphocyte line (CTLL) cells used for the measurement of IL-2 activity. This effect was better observed with suboptimal doses of IL-2 and low concentrations of lipids. The increased IL-2 dependent proliferation is not due to a direct effect of liposomes on CTLL cells but rather to an interaction between IL-2 and liposomes. An interaction between IL-2 and components of fetal calf serum is also demonstrated. The results indicate that liposomes may interfere with IL-2 bioassay but also show the possibility of potentiating IL-2 activity for therapeutic purposes.
The potency of different rabies vaccines was measured via cell mediated immunity (CMI) assessed b... more The potency of different rabies vaccines was measured via cell mediated immunity (CMI) assessed by the production of interleukin-2 (IL-2) by CD4+CD8-lymphocytes. IL-2 production by splenocytes from mice immunized with various vaccines was measured following in vitro stimulation with antigens from different rabies and rabies-related strains. IL-2 production was specific, reproducible and correlated with the vaccine protective activity as determined by the pre-exposure NIH test. Our results suggest that measurement of IL-2 production could be used for the appraisal of rabies vaccine potency.
Objetivo: Rever de forma sistemática as substâncias adjuvantes mais estudadas, utilizadas e as no... more Objetivo: Rever de forma sistemática as substâncias adjuvantes mais estudadas, utilizadas e as novas substâncias; com o intuito de atualizar os conceitos de sua ação por atuarem, juntamente com o antígeno, no sistema imunológico e seu futuro uso em vacinas humanas ou animais.
Rabies is an important viral disease with a great impact on public health due to its extensively ... more Rabies is an important viral disease with a great impact on public health due to its extensively fatal outcome. In endemic areas, rabies may be responsible for over 50,000 human fatalities per year, which is a high number, since a well-established prophylaxis by immunization is available. Despite experimental protocols, no antiviral drugs are currently effective against rabies virus infection. For this reason, antivirals directed to treat human rabies need to be developed. In order to standardize in vitro antiviral screening for rabies virus, we evaluated two methods: the inhibition of cytopathic effect (CPE) and the MTT assay. Anti-rabies activity was only measurable by CPE, while the MTT assay failed to detect antiviral activity in rabies infected McCoy cells. In addition, no significant correlation was observed when comparing both assays. The correlation between the inhibition of CPE and MTT assay was shown when Vero cells were infected with herpes simplex virus type 1 and Hep-2 cells were infected with adenovirus type 5, reinforcing that despite the reliable CPE, McCoy cells infected with rabies cannot be assessed by MTT. In conclusion, these results have shown that the inhibition of CPE should be used for anti-rabies screening in McCoy cells, while the MTT assay was unsuitable in this in vitro system..
Hantaviruses are rodent-borne RNA viruses that cause hemorrhagic fever with renal syndrome (HFRS)... more Hantaviruses are rodent-borne RNA viruses that cause hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). From the first detection of infection in Brazil in 1993 until 2009, 1161 cases of HPS have been reported, with mortality rates of around approximately 40%. Currently, due to the absence of a vaccine or specific antiviral therapy, the only way to reduce mortality by hantavirus infection is a fast and precise diagnosis that allows for supportive clinical health care. To improve the detection of hantavirus infection, we developed monoclonal antibodies (Mabs) against the nucleoprotein (rNDelta85) of the Araucaria hantavirus strain (ARAUV). The specificity of generated Mabs for rNDelta85 was demonstrated by western blot, indirect immunofluorescence and immunohistochemistry. These are the first monoclonal antibodies to be produced and characterized against the South American hantavirus strain, and may be of special interest in the development of diagnos...
Revista do Instituto de Medicina Tropical de São Paulo, 1999
The cellular and humoral immune responses of mice inoculated with rabies virus and treated with t... more The cellular and humoral immune responses of mice inoculated with rabies virus and treated with the Bacillus of Calmette-Guérin, Avridine and Propionibacterium acnes were evaluated in this paper. There was a higher percentage of surviving mice in groups submitted to P. acnes treatment. Lower levels of interferon-gamma (IFN-gamma) were found in infected mice. The intra-pad inoculation test (IPI) was not effective to detect cellular immune response, contrary to the results found in MIF reaction. The survival of mice did not present correlation with the levels of antirabies serum neutralizing (SN) antibodies titers, IFN-gamma concentration and MIF response.
Monoclonal antibodies (mAbs) can be used therapeutically by binding to molecular targets with hig... more Monoclonal antibodies (mAbs) can be used therapeutically by binding to molecular targets with high specificity. Therefore, they have excellent therapeutic applications in ophthalmology. This manuscript presents four aspects of the therapeutic use of mAbs in ophthalmology: the scientific rationale, the unique characteristics of selected mAbs, the current state-of-the-art application, and relevant therapeutic mAbs for future applications in ophthalmology. We identified in the literature various single-agent therapies that inhibit the following targets: tumor necrosis factor (TNF), epithelial growth factor receptor, vascular endothelial growth factor (VEGF) receptor, basic fibroblast growth factor receptor, platelet-derived growth factor, and cluster of differentiation antigens. The roles of all biochemical targets in ocular diseases were evaluated. Current and future mAbs against various cytokines were assessed for the treatment of ocular diseases. The medical literature showed the clinical benefits of mAbs for treating angiogenic and inflammatory ocular diseases. Two anti-VEGF mAbs, bevacizumab and ranibizumab, and three anti-TNF agents, infliximab, etanercept, and adalimumab, control ocular neovascularization and intraocular inflammation. Other mAbs such as rituximab, daclizumab, efalizumab, and alemtuzumab showed positive results in animal and early clinical studies and may represent useful adjuvant therapies for ocular lymphoma or ocular inflammation. Ranibizumab is the only FDA-approved therapy; for other mAbs the so-called off-label application remains the standard. Intravenous administration of mAbs has demonstrated acceptable toxicity profiles, while intraocular injection may decrease the chances of systemic complications and increase the amount of drug available to the retina and choroid. In conclusion, effective clinical use of mAbs in ophthalmology is more commonly seen in the field of angiogenic vitreoretinal and autoimmune inflammatory diseases. The challenge for the future is combining biologic therapies to improve the quality and duration of responses while diminishing side effects. The role of mAbs within ophthalmic treatments will be defined according to future clinical experience and the results of randomized clinical trials.
Using the laboratory mice, Fuenzalida-Palacios mouse brain human rabies vaccine was administered ... more Using the laboratory mice, Fuenzalida-Palacios mouse brain human rabies vaccine was administered in groups of animals previously inoculated with rabies virus and then submitted to treatments with the immunomodulators onco-BCG, avridine and Propionibacterium acnes. Humoral and cellular immune responses were evaluated through the macrophage inhibition factor (MIF), intra-pad inoculation (IPI) and serum neutralization (SN) tests and by the detection of g-interferon (IFN-g). The IPI test was not eective in detecting the response of delayed-type hypersensitivity, contrary to MIF, which showed the immune cellular response. Higher levels of IFN-g were observed in the groups of mice vaccinated and treated with avridine and P. acnes. Although immunomodulating activities have been detected, the use of adjuvants with the Fuenzalida-Palacios type vaccine in mice did not reveal any encouraging results. #
The glycoprotein (G) of rabies virus (RV) is important for virus infectivity and induction of the... more The glycoprotein (G) of rabies virus (RV) is important for virus infectivity and induction of the protective immunity. In this study, the region comprising linear epitopes (residues 179-281, ERA strain), named rGERA179-281, was cloned in frame with a hexahistidine tag coding sequence at its N-terminal end and overexpressed in Escherichia coli Rosetta strain. The expression under transcriptional regulation of T7 promoter yielded insoluble protein aggregates in the form of inclusion bodies. The inclusion bodies were solubilized with 6 M guanidine HCl and the protein was purified to homogeneity under denaturing conditions. Mass spectrometry data confirmed the identity of the protein. The purified protein (13.8 kDa) showed significant reactivity with antibodies present in a therapeutic human rabies immune globulin (HRIG), as demonstrated by immunoblotting analysis. In addition, by in vitro competitive neutralization assay, rGERA179-281 led to a measurable reduction in the ability of HRIG to neutralize rabies virus. These results, along with the good yield obtained, encourage further studies on the more detailed immunological properties of rGER-A179-281, such as the ability to induce rabies virus neutralizing antibodies and the production of anti-G monoclonal antibodies, which together, might be useful for the development of new diagnostic methods.
This work describes the cytopathic effect on cells, cytotoxic action on mice, and antiviral activ... more This work describes the cytopathic effect on cells, cytotoxic action on mice, and antiviral activity of cinnabarin. This substance had no effect on mouse neuroblastoma cells (NA cell, ATCC clone C-1300) at a concentration of 0.31 mg/ml, it was not able to cause toxic effects in mice at concentrations of 1000 mg/kg, and reduced by four times the titers of the rabies virus at concentrations of 0.31 mg/ml.
Ten monoclonal antibodies (MAbs) against rabies virus, including IgG3κ, IgG2aκ, IgMκ, and an IgG2... more Ten monoclonal antibodies (MAbs) against rabies virus, including IgG3κ, IgG2aκ, IgMκ, and an IgG2bκ isotype, were produced and characterized using neutralization, ELISA, immunodot-blot, and immunofluorescence assays. MAb 8D11, which recognized rabies virus glycoprotein, was found to neutralize rabies virus in vitro. When submitted to an immunofluorescence assay, seven MAbs showed different reactivity against 35 Brazilian rabies virus isolates. Three MAbs (LIA 02, 3E6, and 9C7) only failed to recognize one or two virus isolates, whereas MAb 6H8 was found to be reactive against all virus isolates tested. MAbs were also evaluated for their immunoreactivity against fixed rabies virus strains present in human and veterinary commercial vaccines. MAbs LIA 02, 6H8, and 9C7 reacted against all vaccine strains, while the remaining MAbs recognized at least 76% of vaccine strains tested. This research provides a set of MAbs with potential application for improving existing or developing new diagnostic tests and immunoassays.
Murine myeloma cell lines play an important role in different areas of scientific research and ar... more Murine myeloma cell lines play an important role in different areas of scientific research and are essential tools for monoclonal antibody production technology. Thus, it is important to understand the biology of these cell lines in order to provide useful information to various research fronts. The present study aims to perform detailed analyses of surface antigens expressed on three major murine myeloma cell lines extensively used for MAb production. The P3X63Ag8.653 cell line expresses molecules associated with T cell interaction (CD40(low), CD80(low)), as well as antigens related to plasma cell phenotype (CD138(high), CD184(low)). The Sp2/0-Ag14 cell line presents molecules associated with BCR activation and regulation (CD79b(low), CD22(low), CD72(med)), molecules related to T cell interaction (CD40(low), CD80(low)), and markers of plasma cell phenotype (CD138(high), CD184(low)). The NS1 cell line presents all molecules of plasma cell phenotype evaluated in this study (CD184(low), CD138(high), CD38(med)) with low expression of CD72 (CD72(low)), a molecule related to BCR activation. Molecules associated with immune response modulation such as CD23 and CD25, as well as CD117, a marker related to undifferentiated cell phenotype, were not observed in any of the three murine myeloma cell lines evaluated. These data show that in spite of their common origin and function, the immunological profiles differ between P3X63Ag8.653, Sp2/0-Ag14, and NS1 cell lines.
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Papers by Carlos Zanetti