Identification of T cell epitopes on the 33-kDa fragment of Plasmodium yoelii merozoite surface protein 1 and their antibody-independent protective role in immunity to blood stage malarial

Merozoite surface protein 1 (MSP1) of malaria parasites undergoes proteolytic processing at least twice before invasion into a new RBC. The 42-kDa fragment, a product of primary processing, is cleaved by proteolytic enzymes giving rise to MSP1(33), which is shed from the merozoite surface, and MSP1(19), which is the only fragment carried into a new RBC. In this study, we have identified T cell epitopes on MSP1(33) of Plasmodium yoelii and have examined their function in immunity to blood stage malaria. Peptides 20 aa in length, spanning the length of MSP1(33) and overlapping each other by 10 aa, were analyzed for their ability to induce T cell proliferation in immunized BALB/c and C57BL/6 mice. Multiple epitopes were recognized by these two strains of mice. Effector functions of the dominant epitopes were then investigated. Peptides Cm15 and Cm21 were of particular interest as they were able to induce effector T cells capable of delaying growth of lethal P. yoelii YM following adoptive transfer into immuno-deficient mice without inducing detectable Ab responses. Homologs of these epitopes could be candidates for inclusion in a subunit vaccine.

Antibody-dependent enhancement of Murray Valley encephalitis virus virulence in mice

Enhancement of flavivirus infection in vitro in the presence of subneutralizing concentrations of homologous or heterologous antiserum has been well described. However, the importance of this phenomenon in the enhancement of flavivirus infection in vivo has not been established. In order to study antibody- mediated enhancement of flavivirus infection in vivo, we investigated the effect of passive immunization of mice with Japanese encephalitis virus (JE) antiserum on the outcome of infection with Murray Valley encephalitis virus (MVE). We show that prior treatment of mice with subneutralizing concentrations of heterologous JE antiserum resulted in an increase in viraemia titres and in mortality following challenge with wild-type MVE. Our findings support the hypothesis that subneutralizing concentrations of antibody may enhance flavivirus infection and virulence in vivo. These findings are of potential importance for the design of JE vaccination programs in geographic areas in which MVE co-circulates. Should subneutralizing concentrations of antibody remain in the population following JE vaccination, it is possible that enhanced disease may be observed during MVE epidemics.

IL-10 Mediates Suppression of the CD8 T Cell IFN-γ Response to a Novel Viral Epitope in a Primed Host

Priming to Ag can inhibit subsequent induction of an immune response to a new epitope incorporated into that Ag, a phenomenon referred to as original antigenic sin. In this study, we show that prior immunity to a virus capsid can inhibit subsequent induction of the IFN-gamma effector T cell response to a novel CD8-restricted antigenic epitope associated with the virus capsid. Inhibition does not involve Ab to the virus capsid, as it is observed in animals lacking B cells. CD8-restricted virus-specific T cell responses are not required, as printing to virus without CTL induction is associated with inhibition. However, IL-10(-/-) mice, in contrast to IL-10(+/+) mice, generate CD8 T cell and Ab responses to novel epitopes incorporated into a virus capsid, even when priming to the capsid has resulted in high titer Ab to the capsid. Furthermore, capsid-primed mice, unable to mount a response to a novel epitope in the capsid protein, are nevertheless able to respond to the same novel epitope delivered independently of the capsid. Thus, inhibition of responsiveness to a novel epitope in a virus-primed animal is a consequence of secretion of IL-10 in response to presented Ag, which inhibits local generation of new CD8 IFN-gamma-secreting effector T cells. Induction of virus- or tumor Ag-specific CD8 effector T cells in the partially Ag-primed host may thus be facilitated by local neutralization of IL-10.

Epitope map for a growth hormone receptor agonist monoclonal antibody, MAb 263

Monoclonal antibody (MAb) 263 is a widely used monoclonal antibody that recognizes the extracellular domain (ECD) of the GH receptor. It has been shown to act as a GH agonist both in vitro and in vivo, and we report here that it must be divalent to exert its effect on the full-length receptor. To understand the mechanism of its agonist action, we have determined the precise epitope for this antibody using a novel random PCR mutagenesis approach together with expression screening in yeast. A library of 5200 clones of rabbit GH receptor ECD mutants were screened both with MAb 263 and with an anticarboxy-tag antibody to verify complete ECD expression. Sequencing for clones that expressed complete ECD but were not MAb 263 positive identified 20 epitope residues distributed in a discontinuous manner throughout the ECD. The major part of the epitope, as revealed after mapping onto the crystal structure model of the ECD molecule, was located on the side and upper portion of domain 1, particularly within the D - E strand disulfide loop 79 - 96. Molecular dynamics docking of an antibody of the same isotype as MAb 263 was used to dock the bivalent antibody to the 1528-Angstrom(2) epitope and to visualize the likely consequences of MAb binding. The minimized model enables the antibody to grasp two receptors in a pincer-like movement from opposite sides, facilitating alignment of the receptor dimerization domains in a manner similar to, but not identical with, GH.

Detection of anti-Giardia lamblia serum antibody among children of day care centers

OBJETIVES: To detect anti-Giardia lamblia serum antibodies in healthy children attending public day care centers and to assess serological tests as tools for estimating the prevalence of G. lamblia in endemic areas. METHODS: Three separate stool specimens and filter paper blood samples were collected from 147 children ranging from 0 to 6 years old. Each stool sample was processed using spontaneous sedimentation and zinc sulfate flotation methods. Blood samples were tested by indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA) for Giardia IgG. RESULTS AND CONCLUSIONS: Of 147 individuals tested, 93 (63.3%) showed Giardia cysts in their feces. Using IIF and ELISA, serum antibodies were detected in 93 (63.3%) and 100 (68%) samples , respectively. Sensitivity of IIF and ELISA was 82% and 72%, respectively. However, ELISA revealed to be less specific (39%) than IIF (70%). IIF also showed a higher concordance with microscopic examination than ELISA.

An electrochemical deamidated gliadin antibody immunosensor for celiac disease clinical diagnosis

The first electrochemical immunosensor (EI) for the detection of antibodies against deamidated gliadin peptides (DGP) is described here. A disposable nanohybrid screen-printed carbon electrode modified with DGP was employed as the transducer's sensing surface. Real serumsampleswere successfully assayed and the results were corroborated with an ELISA kit. The presented EI is a promising analytical tool for celiac disease diagnosis.

Seroprevalence of Toxoplasma gondii IgG antibody in HIV/AIDS-infected individuals in Maputo, Mozambique

OBJECTIVE To analyze the prevalence of IgG antibodies to Toxoplasma gondii in patients infected with HIV/AIDS and the association of demographic and social variables. METHODS Descriptive cross-sectional study that included the analysis of sociodemographic data and laboratory findings of 200 patients infected with HIV/AIDS treated in a laboratory unit in Maputo, Mozambique, in 2010. Individual data for all participants were collected with a self-administered questionnaire. Plasma samples were tested for IgG testing of anti- T. gondii using hemagglutination for the analysis of antibodies. RESULTS The seroprevalence of IgG anti- T. gondii was 46.0% (95%CI 39.2;52.9), 39.3% (95%CI 29.5;50.0) in men and 50.9% (95%CI 41.9;59.8) in women, with no difference between sex (OR 1.30; 95%CI 0.95;1.77; p = 0.12). Ages ranged from 10 to 60 years, with a higher prevalence of infection in older age groups, but with no significant difference between them. Regularly consuming cattle meat (OR 1.74; 95%CI 1.04;2.89, p = 0.05), breeding cats/dogs (OR 6.18; 95%CI 3.60;10.62, p < 0.000) and having regular contact with soil (OR 3.38; 95%CI 2.19;5.21; p < 0.000) were significantly associated with risk of latent infection. CONCLUSIONS Toxoplasmosis is an infection with high prevalence in Mozambique. Cultural and behavioral aspects increase the risk. Toxoplasmosis can be responsible in our environment by the great burden of morbidity and mortality associated with meningoencephalic injuries in patients with HIV/AIDS.

Analysis of glycosylation heterogeneity in antibody production by Pichia pastoris

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia

Acute Hepatitis B in a patient previously positive for antibody to surface antigen (anti-HBs) determined by radioimmunoassay: case report and review of the literature

The determination of anti-HBs as a screening test before vaccination has been advisable in order to encounter immune individuals that don't need to receive vaccine protection. A case-report is presented and three other cases are reviewed from the literature. Anti-HBs was positive in these health-care personnels that developped typical acute B hepatitis. Different subtyping involving the d/y determinants were found in the first case, but false-positive anti-HBs even with high titres, determined by RIA, were found in the other cases. Concomitant determination of anti-HBc or absence of screening tests seem to be more reasonable policies until a low-cost and risk-free vaccine is produced.

Prevalence of antibody to hepatitis B virus in an urban population of northeast Brazil

A sample of 1,288 inhabitants of Salvador, Bahia, Brazil, were submitted to the determination of anti-HBs using radioimmunoassay procedure, and analysed according to age, sex and income. Overall prevalence of anti-HBs was 11,8%. ranging from 6,7% among children aged less than three years old to 26,1% among those aged 30 years and older. Males presented prevalence of anti-HBs similar to female individuals, and those with a higher income showed frequencies of anti-HBs greater than those with a lower income level. The following conclusions were drawn: The high prevalence of anti-HBs observed among children suggests early contact with hepatitis B virus, possibly due to vertical transmission and intrafamiliar dissemination of the disease; the frequency of anti-HBs increases with age; the lower prevalence of anti-HBs among those with low income suggests that this group may present higher prevalence of carriers of the hepatitis B virus surface antigen.

Antibody response to heat-inactivated hepatitis B vaccine (CLB-3mg) in hemodialysis patients and occupational risk personnel: a one year follow-up

Immune response against hepatitis B vaccine (CLB 3mg) was evaluated in 59 hemodialysis patients and 20 occupational risk personnel. Seroconversion was induced in 52.5% and 70.0% respectively. Twelve months after the first dose, 37.5% of patients and 60.0% of occupational risk personnel had detectable anti-HBs level. Antibody level was expressed in sample ratio units (SRU). Considering only the responders, in the patients group 38.7% had a low anti-HBs response (2.1-9.9 SRU) 32.3% a medium response (10-99.9 SRU) and 29.0% a high response (>100 SRU) while in occupational risk personnel these values were 14.3%, 64.3% and 21.4% respectively. The authors suggest the use of HBV vaccines with more elevated HBsAg concentration or a reinforced immunization schedule to improve the anti-HBs response not only for patients but also for healthy persons.

Immunofluorescent antibody to rotavirus and antigen excretion in infantile acute diarrhoea

A serological study was carried out to evaluate the causal relationship of acute diarrhoea and rotavirus in children under five years of age. The rotaviral infection was demonstrated in paired sera by determining the antibody titers by immunofluorescence test (IF). Bovine rotavirus-infected MA-104 cell culture was used as substratum for IF. Out of 80 paired sera it was shown that 23 (28.75%) presented seroconversion, 19 (23.75%) samples showed a twofold increase in their titers and 38 (47.5%) had no increase in rotavirus antibody. This result is discussed on the light of previously obtained results on viral antigen detection by counterimmunoelectrosmophoresis (CIEOP).

Peroxidase antibody test for mucocutaneous leishmaniasis serology performance indexes and comparison with a fluorescent antibody test: Comparação com o desempenho da reação de imunofluorescência

Performance indexes of the peroxidase antibody test were compared to that of the fluorescent antibody test. The peroxidase antibody test had a statistically higher sensitivity and negative predictive value and a higher efficiency than the fluorescent antibody test but its specificity and positive predictive value were within the 95% confidence limits for the values found for the fluorescent antibody test. Such differences did not change when Chagas' disease and visceral leishmaniasis sera were included in index calculations. Statistical analysis showed that the two tests have a substantial degree of agreement but the immunofluorescent test had a specificity index and a positive predictive value equal to 100.0% when Chagas' disease and visceral leishmaniasis sera were not included in the calculations of the performance index; in this instance, a positive test result equals a disclosure of the disease attribute due to the inexistence of false positive results. The enzyme/ protein ratio of the peroxidase conjugate, resulting in heavy or light-labeled conjugates may pose technical problems to its use in serology tests.