Identificação de epítopos de célula B na glicoproteína-E do envelope do vírus dengue sorotipo 3 / Identification of B-cell epitopes in the envelope glycoprotein of dengue virus type 3

As infecções pelo vírus dengue têm se tornado um problema crescente de Saúde Pública em regiões tropicais e subtropicais do mundo. O vírus pertence à família Flaviviridae com quatro sorotipos antigenicamente distintos (DENV-1 a DENV-4). Uma possível estratégia para evitar a patogenia associada com uma vacina para o dengue (que deve ser tetravalente), seria a construção de uma vacina quimérica composta de epítopos críticos selecionados dos quatro sorotipos. A maioria dos epítopos envolvidos na neutralização do vírus está presente na glicoproteína E do envelope, que é a maior proteína de superfície da partícula viral. O objetivo deste trabalho foi identificar epítopos de célula B na glicoproteína E do vírus dengue sorotipo 3. Para o mapeamento de epítopos imunodominantes, noventa e cinco peptídeos (15-mers cada, sobreposição de 10) foram sintetizados (Synpep, California-USA), a partir da sequência de 490 aminoácidos da glicoproteína E do envelope do DENV-3, de cepa circulante no Brasil. Estes peptídeos foram testados por ELISA contra um pool de soros de pacientes positivos e negativos para dengue, coletados durante a fase de convalescença da infecção por DENV-3. Os resultados mostraram que os soros de humanos reagiram com onze, dos noventa e cinco peptídeos testados, distribuídos em 5 regiões com aminoácidos na posições 51-65 (peptídeo 11), 71-90 (peptídeos 15 e 16), 131-170 (peptídeos 27, 28, 29, 30, 31 e 32), 196-210 (peptídeo 40) e 246-260 (peptídeo 50). A análise da curva ROC mostrou que, dentre os peptídeos identificados, nove seriam capazes de diferenciar entre pacientes com DENV-3 de pacientes não-dengue e três capazes de diferenciar a infecção por DENV-3 daquelas por outros sorotipos virais (DENV-1 e DENV-2). Os epítopos imunodominantes aqui descritos, junto com outros epítopos bem documentados, são potencialmente relevantes para o desenho de uma vacina para o vírus dengue e para o desenvolvimento de kits de diagnóstico específicos.

The specificity of antibody responses in cattle naturally exposed to Fasciola hepatica.

Fasciola hepatica causes significant morbidity and mortality in dairy cattle in the Andean region of Cajamarca, Peru, where prevalence of infection of up to 78% has been reported. ELISA and Western blot analyses were used to characterise antibody responses in dairy cattle to adult F. hepatica to excretory-secretory (E/S), somatic (SO) and surface (SU) antigens. Three groups of dairy cattle - calves, heifers and adult cows - naturally exposed to F. hepatica in this region, were monitored every 2 months over a 2-year period. Calves, heifers and adult cows all had antibodies which recognised a 28kDa protein in the SO preparation, whereas only adult cows had antibodies that recognised a 28kDa protein in E/S products. All three groups of cattle responded to a 60-66kDa group of proteins in E/S and SU preparations and a 17kDa antigen in SO products was recognised by antibodies from cows and heifers but not calves. The total antibody response to E/S antigens measured by ELISA, increased over time in calves and remained constantly high over the 2-year period in all three groups of cattle. Slight fluctuations in the antibody response occurred in the group of heifers and cows coinciding with seasonal changes in the level of challenge.

Identification of immunodominant antigens by immunoelectrotransfer in hydatid fluid.

Identification and characterization of immunodominant antigens in hydatid fluid was performed by electrophoresis in polyacrylamide gels (SDS PAGE) followed by immunoelectrotransfer (Western Blot). The studies were performed in sera of 23 patients with surgically confirmed hydatid disease, 12 patients with clinical suspicion of the infection and positive serology according to conventional serology (double diffusion with detection of are 5 and ELISA test), 28 healthy subject and 23 patients with parasitic infections different from hydatidosis. The results showed 7 antigenic bands located between 8 and 120 kDa, two immunodominant bands (MW 8 and 12 kDa) were recognized by the sera of patients suffering from hydatid disease and those with positive serology. Two additional bands were detected by the sera of healthy subjects and by the samples of patients presenting cysticercosis. It is concluded that the antigens with molecular weights of 8 and 12 kDa. would be those of major diagnostic value, while those of 32 and 60 kDa are nonspecific.

Strongyloides stercoralis: characterization of immunodiagnostic larval antigens

Forty-one-, 31-, and 28-kDa proteins of strongyloides stercoralis filariform larvae have previously been demonstrated to be sensitively and specifically recognized by serum IgG in individuals with strongyloidiasis. Characteristics of these proteins, their immunodominant epitopes, and reactive antibodies are described here. The proteins are soluble is aqueous as well as detergent extracts. The immunodominant epitopes are present in S. stercoralis but not in S. cebus or S. ratti. Epitopes on the three proteins are not shared, as determined by cross-absorption of serum with each of the size components on nitrocellulose. In most sera from strongyloidiasis patients there was reactivity to each of the proteins by IgG1 and IgG4, but reactivity by IgG2 or IgG3 was detectable only in a minority. A rabbit antiserum raised to a 41-kDa size fraction of S. stercoralis larvae reacted against a doublet of 41-kDa which was distinct from the immunodiagnostic 41-kDa protein.(AU)

Recognition of B and T cell epitopes by cattle immunized with a synthetic peptide containing the major immunogenic site of VP1 FMDV 01 Campos.

The precise location of B and T cell epitopes have been established in a peptide containing the major immunogenic site (residues 135-160) of FMDV strain 01 Campos (01C) VP1. The peptide (p135-160), administered free or conjugated to bovine serum albumin, induced complete protection in guinea pigs and a strong neutralizing antibody (NAb) response in cattle. Using a set of partially overlapping peptides it was shown that although several B cell epitopes were distributed along the p135-160, the residues responsible for the induction of NAb were restricted to the amino acids 135-144. In addition, bovines immunized with the p135-160 showed a strong proliferative response to this peptide but a very poor response against purified virus. Two T-epitopes were located in the 135-160 sequence: one on the region 135-144 and the other in the region 150-160. The immunodominance of these two T cell epitopes was confirmed in cattle immunized with inactivated virus vaccines.

Comparative characterization of antigenic epitopes in the immunodominant region of the protein encoded by open reading frame 3 in Burmese and Mexican strains of hepatitis E virus.

To analyse the effect of strain-specific sequence variation on the antigenic properties of the protein encoded by the open reading frame 3 (ORF 3) of hepatitis E virus (HEV), two sets of short overlapping peptides spanning amino acids 91 to 123 of this protein from Burmese and Mexican strains were synthesized and tested with sera obtained from outbreaks of enterically transmitted non-A, non-B hepatitis in three different regions of the world (Mexico, Turkmenistan and Kenya). The data suggest strain-specific variation in the antigenic reactivity of the ORF 3 protein. The C-terminal region of this protein contains several antigenic epitopes located in the most variable positions. Individual sera were found to interact with different groups of epitopes from each set of peptides. The antigenic epitopes of the Mexican strain appear to be less conformation-dependent than those of the Burmese strain. The most immunoreactive epitope of the ORF 3 protein from the Mexican strain was localized at amino acid positions 95 to 101. The ORF 3 protein of the Burmese strain contains an immunodominant epitope at amino acid positions 112 to 117. Some of these short peptides may be useful for the development of a diagnostic assay to discriminate between the Burmese and Mexican strains.