RESUMO
This study compared the efficacies of two N-methylglucomine antimoniate (MA) dose regimens for treating macaques with Leishmania braziliensis-induced chronic skin disease. Whereas all animals treated with the full dose (20 mg MA/kg/day) were cured, 50% of the monkeys receiving a low-dose regimen (5 mg MA/kg/day) relapsed. The antimony concentrations in macaque plasma and tissue samples were greater in the full-dose group than in that receiving a subtherapeutic MA regimen. Our data also suggest the presence of drug-induced hepatic pathology.
Assuntos
Antiprotozoários/uso terapêutico , Leishmaniose Cutânea/tratamento farmacológico , Meglumina/uso terapêutico , Compostos Organometálicos/uso terapêutico , Animais , Antimônio/sangue , Antiprotozoários/administração & dosagem , Rim/parasitologia , Rim/patologia , Leishmania braziliensis , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Fígado/parasitologia , Fígado/patologia , Macaca mulatta , Meglumina/administração & dosagem , Antimoniato de Meglumina , Compostos Organometálicos/administração & dosagem , Baço/parasitologia , Baço/patologiaRESUMO
Among the flaviviruses, dengue, with its four serotypes, has spread throughout the tropics. The most advanced vaccines developed so far include live attenuated viruses, which have been tested in humans but none has been licensed. Preclinical testing of dengue vaccine candidates is performed initially in mice and in nonhuman primates. In the latter the main criteria used to assay protection are neutralizing antibodies elicited by the vaccine candidate and the magnitude and duration of peripheral viremia upon challenge of previously immunized animals. Towards the identification of wild-type viruses that could be used in challenge experiments a total of 31 rhesus monkeys were inoculated subcutaneously of wild dengue types 1, 2, and 3 viruses. The viremia caused by the different viruses was variable but it was possible to identify dengue viruses useful as challenge strains.
Assuntos
Vírus da Dengue , Dengue/virologia , Viremia/virologia , Animais , Chlorocebus aethiops , Dengue/prevenção & controle , Vacinas contra Dengue/uso terapêutico , Vírus da Dengue/classificação , Vírus da Dengue/imunologia , Vírus da Dengue/patogenicidade , Modelos Animais de Doenças , Feminino , Humanos , Macaca mulatta/virologia , Masculino , Células Vero/virologiaRESUMO
Among the flaviviruses, dengue, with its four serotypes, has spread throughout the tropics. The most advanced vaccines developed so far include live attenuated viruses, which have been tested in humans but none has been licensed. Preclinical testing of dengue vaccine candidates is performed initially in mice and in nonhuman primates. In the latter the main criteria used to assay protection are neutralizing antibodies elicited by the vaccine candidate and the magnitude and duration of peripheral viremia upon challenge of previously immunized animals. Towards the identification of wild-type viruses that could be used in challenge experiments a total of 31 rhesus monkeys were inoculated subcutaneously of wild dengue types 1, 2, and 3 viruses. The viremia caused by the different viruses was variable but it was possible to identify dengue viruses useful as challenge strains.
Assuntos
Humanos , Animais , Masculino , Feminino , Vírus da Dengue/classificação , Vírus da Dengue/patogenicidade , Viremia/virologia , Chlorocebus aethiops , Modelos Animais de Doenças , Macaca mulatta/virologia , Células Vero/virologiaRESUMO
Dengue virus, a mosquito-borne flavivirus, is one of the most formidable public health threats in tropical and subtropical regions. As yet, there is no licensed vaccine to protect against the disease. A chimeric yellow fever (YF) 17D/dengue (DEN) type 1 virus was constructed by replacing the pre-membrane and envelope genes of YF 17D virus with those from DEN 1 VeMir95 virus, a Venezuelan isolate. The chimeric YF 17D/DEN 1 VeMir95 virus was regenerated from full-length infectious clones stably propagated in Escherichia coli by transfection of Vero cells with in vitro transcribed RNA. The chimeric virus proliferated efficiently in Vero cells ( approximately 6.6 log(10) plaque-forming units/ml). The chimeric virus was not neurovirulent to 3-week-old Swiss Webster mice inoculated by the intracerebral route, in contrast to the YF 17DD vaccine strain that was lethal for 90% of the mice. The YF 17D/DEN 1 virus at Passage 6 was more attenuated for rhesus monkeys than the YF 17DD commercial vaccine after intracerebral inoculation according to the standard neurovirulence test. This virus is a potential candidate to be included in a tetravalent DEN vaccine formulation. The availability of the cloned cDNA allows further structure/function studies on the viral envelope.
Assuntos
Vírus da Dengue/genética , Vírus Reordenados/genética , Vírus da Febre Amarela/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Chlorocebus aethiops , Vacinas contra Dengue , Vírus da Dengue/crescimento & desenvolvimento , Vírus da Dengue/patogenicidade , Genes Virais , Camundongos , Dados de Sequência Molecular , Vírus Reordenados/crescimento & desenvolvimento , Vírus Reordenados/patogenicidade , Recombinação Genética , Transfecção , Vacinas Atenuadas , Células Vero , Proteínas do Envelope Viral/genética , Virulência , Vírus da Febre Amarela/crescimento & desenvolvimento , Vírus da Febre Amarela/patogenicidadeRESUMO
Visceral leishmaniasis (VL) was experimentally induced in rhesus macaques (Macaca mulatta) by intravenously inoculating 2 x 10(7)amastigotes/kg of body weight of Leishmania infantum. The macaques developed a systemic disease showing characteristic features of human VL such as fever, diarrhoea, body weight loss, anaemia, hypergammaglobulinaemia and transient lymphocytosis, as well as lymph node, liver and/or spleen enlargement. Nine weeks after infection, one primate showed pronounced weight loss, became moribund and was euthanized. The necropsy findings included granulomas composed of parasite-containing macrophages, lymphocytes and plasma cells in the liver, spleen and lymph nodes. The remaining macaques had a sustained course of infection but developed a mild-to-moderate illness that subsequently showed evidence of self-cure. Of note, pathological findings included a typical cell-mediated immunity-induced granulomatous reaction that had an effect on the control of parasite replication. All infected monkeys responded with increased production of anti-Leishmania-specific IgG antibodies. Despite the fact that clinical resistance to L. infantum was not consistently associated with a parasite-specific cell-mediated immune response, drug-cured macaques from the primary infection acquired immunity to homologous re-infection. These findings point to the feasibility of using the L. infantum macaque model for pre-clinical evaluation of novel chemotherapeutics or vaccine candidates for human VL.
Assuntos
Modelos Animais de Doenças , Leishmania infantum/imunologia , Leishmaniose Visceral/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Formação de Anticorpos , Antígenos de Protozoários/sangue , DNA de Protozoário/análise , Ensaio de Imunoadsorção Enzimática , Doenças Hematológicas/parasitologia , Imunidade Celular , Imuno-Histoquímica , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico , Macaca mulatta , MasculinoRESUMO
A chimeric yellow fever (YF)-dengue serotype 2 (dengue 2) virus was constructed by replacing the premembrane and envelope genes of the YF 17D virus with those from dengue 2 virus strains of Southeast Asian genotype. The virus grew to high titers in Vero cells and, after passage 2, was used for immunogenicity and attenuation studies in rhesus monkeys. Subcutaneous immunization of naive rhesus monkeys with the 17D-D2 chimeric virus induced a neutralizing antibody response associated with the protection of 6 of 7 monkeys against viremia by wild-type dengue 2 virus. Neutralizing antibody titers to dengue 2 were significantly lower in YF-immune animals than in YF-naive monkeys and protection against challenge with wild-type dengue 2 virus was observed in only 2 of 11 YF-immune monkeys. An anamnestic response to dengue 2, indicated by a sharp increase of neutralizing antibody titers, was observed in the majority of the monkeys after challenge with wild-type virus. Virus attenuation was demonstrated using the standard monkey neurovirulence test. The 17D-D2 chimera caused significantly fewer histological lesions than the YF 17DD virus. The attenuated phenotype could also be inferred from the limited viremias compared to the YF 17DD vaccine. Overall, these results provide further support for the use of chimeric viruses for the development of a new live tetravalent dengue vaccine.
Assuntos
Animais , Masculino , Feminino , Anticorpos Antivirais/biossíntese , Viremia/imunologia , Vírus da Dengue/imunologia , Vírus da Febre Amarela/imunologia , Sequência de Aminoácidos , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Macaca mulatta , Dados de Sequência Molecular , Testes de Neutralização , Recombinação Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Vero , Vírus da Dengue/genética , Vírus da Febre Amarela/genéticaRESUMO
A chimeric yellow fever (YF)-dengue serotype 2 (dengue 2) virus was constructed by replacing the premembrane and envelope genes of the YF 17D virus with those from dengue 2 virus strains of Southeast Asian genotype. The virus grew to high titers in Vero cells and, after passage 2, was used for immunogenicity and attenuation studies in rhesus monkeys. Subcutaneous immunization of naive rhesus monkeys with the 17D-D2 chimeric virus induced a neutralizing antibody response associated with the protection of 6 of 7 monkeys against viremia by wild-type dengue 2 virus. Neutralizing antibody titers to dengue 2 were significantly lower in YF-immune animals than in YF-naive monkeys and protection against challenge with wild-type dengue 2 virus was observed in only 2 of 11 YF-immune monkeys. An anamnestic response to dengue 2, indicated by a sharp increase of neutralizing antibody titers, was observed in the majority of the monkeys after challenge with wild-type virus. Virus attenuation was demonstrated using the standard monkey neurovirulence test. The 17D-D2 chimera caused significantly fewer histological lesions than the YF 17DD virus. The attenuated phenotype could also be inferred from the limited viremias compared to the YF 17DD vaccine. Overall, these results provide further support for the use of chimeric viruses for the development of a new live tetravalent dengue vaccine.
Assuntos
Anticorpos Antivirais/biossíntese , Vírus da Dengue/imunologia , Viremia/imunologia , Vírus da Febre Amarela/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Vírus da Dengue/genética , Feminino , Macaca mulatta , Masculino , Dados de Sequência Molecular , Testes de Neutralização , Recombinação Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Vero , Vírus da Febre Amarela/genéticaRESUMO
Common marmosets (Callithrixjacchus) were orally inoculated with a Brazilian strain (HAF-203) of hepatitis A virus (HAy). Three monkeys were euthanized at postinoculation hours 6, 12 and 24 to investigate the early events of HAV infection. Following others three inoculated and one control marmosets remained throughout the 46 day to evaluation of viral excretion. Different samples were collected to detect sequential presence of HAV RNA by nested reverse transcription-polymerase chain reaction (RT-PCR) in liver, saliva, bile and stools at 6 hours to 461h days postinoculation. Liver tissues were examined by immunofluorescence assay in a confocal laser-scanning microscope for the presence of HAV antigen. HAV RNA was detected in saliva during the course of the study, in bile from 24 hours to 46 days. in stools from 7 to 46 days and liver at 12 hours postinfection. In immunofluorescence of liver stained preparations, viral antigen was present at six hours after inoculation throughout the remainder of the 46-day study. The animals developed histological and biochemical acute hepatitis after second week postinoculation. Spleen, duodenum, and mesenteric lymph nodes specimens were negative for HAV antigens. This study supports the possibility that in Callithrixjacchus orally inoculated with hepatitis A virus the saliva route may be additional way of viral elimination. The viral replication in the liver was responsible for biliary HAV presence and latter HAV detection in fecal samples.
Assuntos
Antígenos Virais/análise , Callithrix , Vírus da Hepatite A/imunologia , Hepatite A/imunologia , Doenças dos Macacos/imunologia , Replicação Viral/imunologia , Animais , Modelos Animais de Doenças , Hepatite A/patologia , Hepatite A/transmissão , Antígenos da Hepatite A , Vírus da Hepatite A/crescimento & desenvolvimento , Vírus da Hepatite A/isolamento & purificação , Fígado/imunologia , Fígado/patologia , Fígado/virologia , Doenças dos Macacos/transmissão , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The yellow fever (YF) 17D virus is one of the most successful vaccines developed to data. Its use has been estimated to be over 400 million doses with an excellent record of safety. In the past 3 years, yellow fever vaccination was intensified in Brazil in response to higher risk of urban outbreaks of the disease. Two fatal adverse events temporally associated with YF vaccination were reported. Both cases had features similar to yellow fever disease, including hepatitis and multiorgan failure. Two different lots of YF 17DD virus vaccine were administered to the affected patients and also to hundreds of thousands of other individuals without any other reported serious adverse events. The lots were prepared from the secondary seed, which has been in continuous use since 1984. Nucleotide sequencing revealed minor variations at some nucleotide positions between the secondary seed lot virus and the virus isolates from patients; these differences were not consistent across the isolates, represented differences in the relative amount of each nucleotide in a heterogeneous position, and did not result in amino acid substitutions. Inoculation of rhesus monkeys with the viruses isolated from the two patients by the intracerebral (ic) or intrahepatic (ih) route caused minimal viremia and no clinical signs of infection or alterations in laboratory markers. Central nervous system histological scores of rhesus monkeys inoculated ic were within the expected range, and there were no histopathological lesions in animals inoculated ih. Altogether, these results demonstrated the genetic stability and attenuated phenotype of the viruses that caused fatal illness in the two patients. Therefore, the fatal adverse events experienced by the vaccinees are related to individual, genetically determined host factors that regulate cellular susceptibility to yellow fever virus. Such increased susceptibility, resulting in clinically overt disease expression, appears to be extremely rare.
Assuntos
Vacina contra Febre Amarela/genética , Febre Amarela/virologia , Vírus da Febre Amarela/genética , Animais , Anticorpos Antivirais/sangue , Brasil , Chlorocebus aethiops , Qualidade de Produtos para o Consumidor , Modelos Animais de Doenças , Feminino , Humanos , Macaca mulatta , Masculino , Fenótipo , Análise de Sequência de DNA , Vacinação , Células Vero , Viremia , Febre Amarela/prevenção & controle , Vacina contra Febre Amarela/efeitos adversos , Vírus da Febre Amarela/crescimento & desenvolvimento , Vírus da Febre Amarela/fisiologiaRESUMO
Callithrix jacchus is considered a reliable animal model for hepatitis A virus (HAV) infection. All three HAV orally inoculated marmosets developed hepatitis - the infection was monitored by continuous virus shedding, high levels of serum enzyme alanine aminotransferase, specific antibody and seroconversion 3-6 weeks after HAV inoculation. HAV antigen was detected in liver by immunofluorescence 4 days post inoculation (PI) and onwards. To gain insight into the biological role of inducible nitric oxide synthase (iNOS) during immune-related acute liver injury the enzyme was searched in frozen biopsies: immunofluorescent labeling was found in the cytoplasm of liver cells mainly Kupffer's cells and spleen macrophages (CD68+) starting 11 days PI with maximum intensity on the fifth to sixth week PI. Necroinflammatory liver lesions characteristic of viral hepatitis were also observed at 10 days PI with maximum severity at 4 to 6 weeks PI. Furthermore, T lymphocytes (CD2+) were raised at this time point. No difference was evident in the frequency of B lymphocytes (CD20+). Therefore, iNOS expression preceded necroinflammatory liver lesion and maximal immunofluorescence reaction was coincident with tissue injury, supporting the hypothesis that NO contributes to hepatic cytotoxic mechanism but also to virus clearance. The concomitant rise in T-lymphocyte population may suggest a role for these cells in this and/or other independent HAV-induced pathological changes.