RESUMO
Bacterial nasal colonization is common in many mammals and Staphylococcus represents the main pathogen isolated. Staphylococcus nasal carriage in humans constitutes a risk factor for Staphylococcus infections pointing out the need for animal experimentation for nasal colonization studies, especially for vaccine development. A limitation in addressing this hypothesis has been a lack of appropriate animal model. Murine models do not mimic human nasal colonization studies. Non-human primates (NHP) remain the best classical models for nasal colonization studies. In this study, we analyzed nasal colonization between two species of Old World monkeys (cynomolgus and rhesus) and a New World monkey (squirrel monkey) from breeding colony at Fiocruz (Brazil). Sixty male and female NHP with the average age of 1-21 years old, comprising twenty animals of each species, were analyzed. Nine different Staphylococcus species (S. aureus, S. cohnii, S. saprophyticus, S. haemolyticus, S. xylosus, S. warneri, S. nepalensis, S. simiae, and S. kloosi) were identified by MALDI-TOF and 16S rRNA gene sequence analyses. Antibiotic resistance was not detected among the isolated bacterial population. S. aureus was the main isolate (19 strains), present in all species, predominant in cynomolgus monkeys (9/20) and squirrel monkeys (7/20). spa typing was used to examine the clonal structure and genetic profile of Staphylococcus aureus isolates. Eight (8) spa types were identified among the S. aureus strains. A major cluster was identified, corresponding to a new spa type t20455, and no spa types found in this study were seen before in Brazil.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Masculino , Humanos , Feminino , Animais , Camundongos , Staphylococcus/genética , Staphylococcus aureus/genética , RNA Ribossômico 16S/genética , Nariz , Infecções Estafilocócicas/microbiologia , Primatas/genética , Staphylococcus aureus Resistente à Meticilina/genética , Portador Sadio/epidemiologia , Mamíferos/genéticaRESUMO
The live attenuated yellow fever (YF) vaccine was developed in the 1930s. Currently, the 17D and 17DD attenuated substrains are used for vaccine production. The 17D strain is used for vaccine production by several countries, while the 17DD strain is used exclusively in Brazil. The cell passages carried out through the seed-lot system of vaccine production influence the presence of quasispecies causing changes in the stability and immunogenicity of attenuated genotypes by increasing attenuation or virulence. Using next-generation sequencing, we carried out genomic characterization and genetic diversity analysis between vaccine lots of the Brazilian YF vaccine, produced by BioManguinhos-Fiocruz, and used during 11 years of vaccination in Brazil. We present 20 assembled and annotated genomes from the Brazilian 17DD vaccine strain, eight single nucleotide polymorphisms and the quasispecies spectrum reconstruction for the 17DD vaccine, through a pipeline here introduced. The V2IDA pipeline provided a relationship between low genetic diversity, maintained through the seed lot system, and the confirmation of genetic stability of lots of the Brazilian vaccine against YF. Our study sets precedents for use of V2IDA in genetic diversity analysis and in silico stability investigation of attenuated viral vaccines, facilitating genetic surveillance during the vaccine production process.
RESUMO
Antibodies that block interaction of immune checkpoint receptors with its ligands have revolutionized the treatment of several cancers. Despite the success of this approach, the high cost has been restricted the use of this class of drugs. In this context, the development of biosimilar can be an important strategy for reducing prices and expanding access after patent has been dropped. Here, we evaluated the use of HEK293 cells for transient expression of an immune checkpoint-blocking antibody as a first step for biosimilar development. Antibody light and heavy chain genes were cloned into pCI-neo vector and transiently expressed in HEK293 cells. The culture supernatant was then subjected to protein A affinity chromatography, which allowed to obtain the antibody with high homogeneity. For physicochemical comparability, biosimilar antibody and reference drug were analyzed by SDS-PAGE, isoelectric focusing, circular dichroism and fluorescence spectroscopy. The results indicated that the both antibodies have a high degree of structural similarity. Lastly, the biosimilar antibody binding capacity to target receptor was shown to be similar to reference product in ELISA and flow cytometry assays. These data demonstrate that the HEK293 system can be used as an important tool for candidate selection and early development of biosimilar antibodies.
Assuntos
Anticorpos Monoclonais/farmacologia , Medicamentos Biossimilares/farmacologia , Inibidores de Checkpoint Imunológico/farmacologia , Proteínas de Checkpoint Imunológico/genética , Cadeias Pesadas de Imunoglobulinas/farmacologia , Cadeias Leves de Imunoglobulina/farmacologia , Anticorpos Monoclonais/biossíntese , Afinidade de Anticorpos , Especificidade de Anticorpos , Medicamentos Biossimilares/metabolismo , Cromatografia de Afinidade , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Inibidores de Checkpoint Imunológico/imunologia , Proteínas de Checkpoint Imunológico/imunologia , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Leves de Imunoglobulina/biossíntese , Focalização IsoelétricaRESUMO
Leptospirosis presents a complex and dynamic epidemiology. Bovine leptospirosis has been described as a major infectious disease impairing reproductive efficiency. Although infections by Leptospira interrogans, L. santarosai and L. borgpetersenii are frequently reported in cattle, the presence of L. noguchii in these animals should not be neglected. In this study, we describe serological (MAT) and molecular characterization (rrs and secY gene sequencing, multilocus sequence typing [MLST] and pulsed-field gel electrophoresis [PFGE]) of eight L. noguchii strains obtained from slaughtered cows. Intraspecific genetic diversity was evaluated, and haplotype networks were constructed based on hosts and geographical localizations. Strains were characterized as belonging to serogroups Australis, Autumnalis and Panama, and molecular characterization showed a high heterogeneity of these strains. Ten different STs were found (including nine new STs and 39 novel alleles) as well as nine different pulsotypes. Two clonal complexes were found. Phylogenetic trees based on secY locus and concatenated MLST loci showed two main clusters, with sequences from the present study included in the first. In general, there was no relationship between the geographical origin and the secY phylogenetic clusters, as well as between secY phylogenetic clusters and serogroups. Molecular diversity indexes confirmed a high variability (H > 0.8). This high intraspecific variation observed may be related to differences in virulence, pathogenicity and antigenicity or even adaptability of the strains. In addition, haplotype networks clearly demonstrated the circulation of genotypes between humans and animals, confirming the zoonotic potential. The present study provides relevant data for the study of leptospirosis in the One Health context, where human, animal and environmental health is closely connected.
Assuntos
Doenças dos Bovinos/epidemiologia , Leptospira/genética , Leptospirose/veterinária , Saúde Única , Animais , Técnicas de Tipagem Bacteriana/veterinária , Bovinos , Doenças dos Bovinos/microbiologia , Eletroforese em Gel de Campo Pulsado/veterinária , Feminino , Genótipo , Humanos , Leptospira/classificação , Leptospira/imunologia , Leptospira/patogenicidade , Leptospirose/epidemiologia , Leptospirose/microbiologia , Epidemiologia Molecular , Tipagem de Sequências Multilocus/veterinária , Panamá/epidemiologia , Filogenia , Sorogrupo , Virulência , ZoonosesRESUMO
Yellow Fever (YF) is an acute viral hemorrhagic disease prevalent mainly in Africa and Americas, with 20-60% fatality rate in severe forms. Currently, antiviral drugs for the infection are not available, reinforcing the importance of vaccination in resident populations and travelers. Manufactured in 7 different countries, the YF vaccine was first created in 1937 and two substrains are used for production, 17DD and 17D-204. The vaccine produced in Bio-Manguinhos/Brazil uses 17DD substrain and more than 160 million doses have been exported to over 74 countries. The World Health Organization (WHO) recommends that new seed- and working-lots should have the viral genome sequenced in order to check vaccine genetic stability. The aim of this study was to develop and standardize a Sanger-based sequencing protocol for the genetic monitoring of the Brazilian 17DD vaccine. We designed 54 oligos to access the complete YF vaccine genome by RT-PCR and sequencing approach. After protocol standardization, we tested 45 vaccine lots and the corresponding secondary and working seed lots. All 45 lots presented 100% nucleotide identity to each other and to the seed lots. We also detected 2 heterogeneous positions at nucleotides 4523 (C/T) and 6673 (C/T) that may indicate a quasispecies diversity of YF 17DD strain. When compared to the Brazilian GenBank sequence YFU17066, the Brazilian 17DD vaccine presented 6 silent mutations. By applying the sequencing methodology to two YF 17D-204 strains, we showed that our method can also be used to sequence different YF vaccine virus. In summary, we have developed a robust method for the genetic monitoring of YF vaccines, which has been successfully applied in Bio-Manguinhos since 2009 and could also be used by other manufacturers for YF17D-based vaccines. There were no genetic variation in the Brazilian tested lots, highlighting the safety, production consistency and, more importantly, the genetic stability of Bio-Manguinhos' YF vaccine in the last 3 decades.
Assuntos
Controle de Qualidade , Vacinas Virais/normas , Sequenciamento Completo do Genoma , Vacina contra Febre Amarela/normas , Febre Amarela/prevenção & controle , Vírus da Febre Amarela/genética , Brasil , Bases de Dados de Ácidos Nucleicos , Genoma Viral , Humanos , Mutação , Vacinas Virais/genética , Organização Mundial da Saúde , Febre Amarela/imunologia , Vacina contra Febre Amarela/genética , Vírus da Febre Amarela/imunologiaRESUMO
PURPOSE: Leptospira interrogans serogroup Icterohaemorrhagiae strains have been described as causing disease in both humans and animals and as being present worldwide. Icterohaemorrhagiae and Copenhageni serovars are known to cause severe disease in their hosts, and zoonotic outbreaks have been described. The genetic similarity among the strains of these serovars is known. However, it has not yet been demonstrated whether major clonal subpopulation in humans, strain Fiocruz L1-130-like, can circulate among other hosts. METHODOLOGY: We performed genetic characterization of Brazilian serogroup Icterohaemorrhagiae strains of dog and rat origin by secY sequencing, variable-number tandem-repeat, multilocus sequence type and multi-spacer typing analysis. RESULTS: The strains were found to be identical among themselves and to strain Fiocruz L1-130. We suggest that the major strain of L. interrogans serogroup Icterohaemorrhagiae, Fiocruz L1-130, is widely distributed in Brazil in different hosts with substantial zoonotic potential. CONCLUSION: Understanding the circulation of strain Fiocruz L1-130 is important for the implementation of appropriate control measures. Its circulation highlights the need to treat leptospirosis caused by L. interrogans serogroup Icterohaemorrhagiae as a zoonosis that acts in the human-animal-environment interface, as per the One Health approach.
Assuntos
Doenças do Cão/microbiologia , Leptospira interrogans/isolamento & purificação , Leptospirose/veterinária , Doenças dos Roedores/microbiologia , Animais , Brasil , Cães , Leptospira interrogans/classificação , Leptospira interrogans/genética , Leptospirose/microbiologia , Repetições Minissatélites , Tipagem de Sequências Multilocus , Filogenia , RatosRESUMO
Leptospirosis in bovines is in majority determined by the host-adapted serovars, mainly Hardjo (types Hardjoprajitno and Hardjobovis), that belong to the serogroup Sejroe. Members of other serogroups as Pomona and Tarassovi have been eventually reported, mainly when outbreaks occurs. Nevertheless, the real role of other strains (non-Hardjo) on determining disease or being transmitted by cattle free of apparent clinical signs of acute infection remains to be elucidated. In that context, the aim of the present study was to investigate the hypothesis that strains of serovars/serogroups other than Hardjo may also be maintained and shed by cattle free of clinical signs. Samples of urine and/or vaginal fluid were collected from 697 bovines from a slaughterhouse located close to Rio de Janeiro, Brazil. Culturing yielded 19 isolates what represents the largest number ever obtained in Brazil on similar studies. These strains were serogrouped and genetically characterized. Fifteen of those were described in other papers and four are first described on the present study. Isolates belong to three different species (Leptospira santarosai, L. alstonii and L. interrogans) and five serogroups (Sarmin, Tarassovi, Shermani, Grippotyphosa and Sejroe). The majority (84.2%) of the isolates belongs to the species L. santarosai, the most prevalent species on cattle in the studied region. Non-Hardjo (non-Sejroe) strains represent 57.9% of the isolates, what indicates an unexpected high diversity of serogroups obtained from these cattle. This suggest that non-Hardjo (non-Sejroe) strains may also be maintained and shed by cattle and that finding must be considered in the epidemiology and control of the disease.
Assuntos
Doenças dos Bovinos/microbiologia , Leptospira/classificação , Leptospira/isolamento & purificação , Leptospirose/veterinária , Animais , Brasil/epidemiologia , Bovinos , Feminino , Leptospirose/epidemiologia , Leptospirose/microbiologia , SorogrupoRESUMO
The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS.
Assuntos
Baculoviridae/química , Baculoviridae/metabolismo , Vírus da Hepatite A/química , Proteínas Virais/biossíntese , Baculoviridae/genética , Regulação Viral da Expressão Gênica , Vetores Genéticos , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Solubilidade , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS.
Assuntos
Baculoviridae/química , Baculoviridae/metabolismo , Vírus da Hepatite A/química , Proteínas Virais/biossíntese , Baculoviridae/genética , Regulação Viral da Expressão Gênica , Vetores Genéticos , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Solubilidade , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Leptospirosis is a zoonotic disease of global importance, and has a worldwide distribution. Equine leptospirosis is commonly manifested by recurrent uveitis, reproductive disorders, as abortions, embryonic absorption, stillbirth and the birth of weak foals. The aim of this study was to verify the presence of Leptospira sp or its DNA in genital tract of mares with reproductive problems. A total of 38 mares with reproductive problems were studied. All the mares were sampled for blood (for serology), urine (for culturing and qPCR), vaginal fluid-VF and endometrial biopsy-EB (for culturing, qPCR and indirect immunofluorescence). PCRs products were sequenced for secY gene. Seventeen (44.7%) serum samples were reactive, predominantly against serogroups Australis (76.4%) and Pomona (23.6%). No positive culture was obtained, but DNA was detected by qPCR on urine samples (26.3%), VF (44.7%) and EB (18.4%) collected 2 months or longer following diagnosis of early fetal death and endometritis. Leptospira cell aggregations were visible by indirect immunofluorescence on 57.1% (4/7) EBs and 17.6% (3/17) VFs. A total of 18 amplicons showed interpretable sequences. Out of those 18 amplicons, 15 presented 100% of identity with the species L. interrogans (sv Bratislava and Pomona), while three were L. borgpertersenii. This study suggests the presence of leptospires in the uterus of mares with reproductive problems. Moreover, serology was shown not to be indicated for the diagnosis of presumptive Leptospira infection in early gestation. The most common agent of the genital infection in those mares was L. interrogans, most probably sg Australis.