Infectious minor lymphocyte stimulating (Mls) antigens.

Minor lymphocyte stimulating (Mls) antigens have profound effects on the murine immune system and have been very important for our current understanding of immune tolerance. It has recently been discovered that these Mls antigens are encoded in an open reading frame located in the 3' long terminal repeat of endogenous and infectious mouse mammary tumor viruses (MMTV). In this review we will discuss the effects of a novel infectious MMTV with properties of Mls-1a on the neonatal and adult immune system in comparison to the effects of endogenous Mtv-7 (Mls-1a).

Whole blood assay to access T cell-immune responses to Mycobacterium tuberculosis antigens in healthy Brazilian individuals

The production of interferon gamma (IFNgamma) guarantees effective T cell-mediated immunity against Mycobacterium tuberculosis infection. In the present study, we simply compare the in vitro immune responses to Mycobacterium antigens in terms of IFNg production in a total of 10 healthy Brazilian volunteers. Whole blood and mononuclear cells were cultivated in parallel with PPD, Ag85B, and M. bovis hsp65, and five-days supernatants were harvested for cytokine detection by ELISA. The inter-assay result was that the overall profile of agreement in response to antigens was highly correlated (r² = 0.9266; p = 0.0102). Potential analysis is in current progress to dictate the usefulness of this method to access the immune responses also in tuberculosis patients and its contacts.

Circulating filarial antigen in the hydrocele fluid from individuals living in a bancroftian filariasis area - Recife, Brazil: detected by the monoclonal antibody Og4C3-assay

The purpose of this study was to examine the circulating filarial antigen (CFA) detected by the monoclonal antibody (mAb) Og4C3-ELISA in paired samples of serum and hydrocele fluid from 104 men with hydrocele, living in an endemic area of Wuchereria bancrofti. Nocturnal blood specimens were filtered and examined for microfilariae (MF) and ultrasound was used in order to identify the presence of adult worms (the filaria dance sign - FDS) in the lymphatic vessels of the scrotal area. Four groups were selected according to their parasitological status: group I - 71 MF- and FDS-; group II - 21 MF+ and FDS+; group III - 10 MF- and FDS+ and group IV- 2 MF+ and FDS-. CFA was identified simultaneously (fluid and serum) in 11 (15.5%), 21 (100%), 3 (30%), and 1 (50%) in groups I, II, III, and IV, respectively. In despite of high CFA+ level (antigen Og4C3) units/ml, the Geometrical Mean (GM) = 2696) in the sera of these 36/104 paired samples, when compared to the hydrocele fluid, (GM = 1079), showed a very good correlation between the CFA level in the serum and CFA level in the fluid (r = 0.731). CFA level in the serum of the 23 microfilaremics (groups II and IV) was extremely high (GM = 4189) and was correlated with MF density (r = 0.442). These findings report for the first time the potential alternative use of the hydrocele fluid to investigate CFA using the mAb Og4C3-ELISA.

Study of the antibody response against Mycobacterium tuberculosis antigens in Warao Amerindian children in Venezuela

This study was aimed at investigating alternate methods for serodiagnosis of tuberculosis (TB), which are needed because bacteriologic diagnosis of childhood TB is difficult. A selection of 80 serum and saliva samples were tested from Warao indigenous children under 15 years of age; 34 high TB suspects (28 positive and 6 negative for the tuberculin skin test, TST) and 46 healthy contact children (32 positive and 14 negative for the TST). Several enzyme-linked immunosorbent assay (ELISA) serological tests were developed to test for Mycobacterium tuberculosis-specific antibodies, including serum IgA, IgG, IgE, and secretory IgA (sIgA) in saliva against 3 specific antigens (PPD, HSP60, 38 kDa). Of these, 2 antigens, PPD and 38 kDa, showed significantly higher reactivity. The sensitivity and specificity of these tests for diagnosis remained limited, between 26.5% and 38.2%, and 77.4% and 97%, respectively. Of all the samples studied and combinations realized between all isotypes and antigens combined with 3 isotypes (anti-PPD IgG, IgE, and anti-38kDa sIgA) managed to detect the largest number of patients, showing an improved sensitivity level of 64.7%, although specificity levels dropped to 81.8%. These results were compared with the Omega diagnostics commercial kit results. The commercial kits showed significantly lower reactivity (sensitivity of 20% and 13.33% to Myco G and Complex Plus, respectively) and a specificity of 100%. This study shows that in indigenous populations of Venezuela, where invasive procedures cannot be used to select samples but evaluation with a chest X-ray for radiological studies is available, the combination of 3 specific isotypes may be a useful tool to increase diagnostic accuracy with pulmonary TB in this population, when used together with clinical and epidemiological criteria.

Antileishmanial IgG and IgE antibodies recognize predominantly carbohydrate epitopes of glycosylated antigens in visceral leishmaniasis

The specificity of human antileishmanial IgG and IgE antibodies to glycosylated antigens of Leishmania chagasi was evaluated. An ELISA was performed with soluble leishmanial antigen (SLA) and a panel of 95 sera including samples from patients with subclinical infection (SC) and visceral leishmaniasis (VL), subjects cured of visceral leishmaniasis (CVL), and from healthy individuals from endemic areas (HIEA). Antileishmanial IgG were verified for 18 (40%) of 45 SC subjects (mean absorbance of 0.49 ± 0.17). All nine sera from VL patients had such antibody (0.99 ± 0.21), while 11 (65%) of 17 CVL individuals were seropositive (0.46 ± 0.05). Only three (12%) of 24 HIEA controls reacted in IgG-ELISA. Antileishmanial IgE was detected in 26 (58%) of 45 SC patients (0.35 ± 0.14), and in all VL patients (0.65 ± 0.29). These antibodies were also detected in 13(76%) of 17 CVL subjects (0.42 ± 0.14) while all HIEA controls were seronegative. There was no correlation between antileishmanial IgG and IgE antibody absorbances. Mild periodate oxidation at acid pH of SLA carbohydrates drastically diminished its antigenicity in both IgG and IgE-ELISA, affecting mainly the antigens of 125, 102, 94, and 63 kDa as demonstrated by western immunoblotting.

Identification of immunodominant epitopes of Schistosoma mansoni vaccine candidate antigens using human T cells

Paramyosin and Sm14 are two of the six antigens selected by the World Health Organization as candidates to compose a subunit vaccine against schistosomiasis. Both antigens are recognized by individuals naturally resistant to Schistosoma mansoni infection and induced protective immunity in the murine model. Three Sm14 epitopes and eleven paramyosin epitopes were selected by their ability to bind to different HLA-DR molecules using the TEPITOPE computer program, and these peptides were synthetically produced. The cellular recognition of Sm14 and paramyosin epitopes by peripheral blood mononuclear cells of individuals living in endemic area for schistosomiasis was tested by T cell proliferation assay. Among all Sm14 and paramyosin epitopes studied, Sm14-3 was preferentially recognized by individuals naturally resistant to S. mansoni infection while Para-5 was preferentially recognized by individuals resistant to reinfection. These two peptides represent promising antigens to be used in an experimental vaccine against schistosomiasis, since their preferential recognition by resistant individuals suggest their involvement in the induction of protective immunity.

Evaluation of enzyme-linked immunosorbent assay using crude Leishmania and recombinant antigens as a diagnostic marker for canine visceral leishmaniasis

The performances of ELISA assays with different antigen preparations, such as Leishmania amazonensis or L. chagasi lysates and the recombinant antigens rK-39 and rK-26, were compared using sera or eluates from dried blood collected on filter paper to detect anti-Leishmania antibodies in dogs from a visceral leishmaniasis-endemic area in Brazil. Of 115 IFAT-reactive dogs at 1:40 titre, 106 (92.2%) were positive in parasitological exams (skin and/or spleen). These animals were compared to healthy animals (n = 25), negative for IFAT at a titre of 1:40 and parasitological exams. The sensitivities of crude and recombinant antigens were similar and remarkably high for both sera and eluates (97-100%). Specificity was higher than 96% for sera and eluates for different antigens, except for L. chagasi antigen using eluates (88%). Concordance values among the tests were higher either for sera or eluates (J = 0.95-1.00). High concordances were observed between sera and eluates tested with different antigens (kappa = 0.93-0.97). Crude and recombinant antigens identified different clinical phases of canine leishmaniasis. These results show that eluates could be used in canine surveys to identify L. chagasi infection. Recombinant antigens added little when compared to crude antigen in identifying positive dogs. Cross-reactivity with other diseases whose distribution often overlaps VL-endemic areas is a limitation of crude antigen use however.

Preliminary study of the presence of antibodies against excretory-secretory antigens from protoscoleces of Echinococcus granulosus in dogs with intestinal echinococcosis

The aim of the present study was to analyze the antibody response against excretory-secretory antigens (ES-Ag) from Echinococcus granulosus protoscoleces, using sera from dogs infected with E. granulosus and other helminths. ES-Ag were obtained from the first 50 h maintenance of protoscoleces in vitro. Immunochemical characterization was performed by immunoblotting with sera from dogs naturally infected with E. granulosus (n = 12), sera from dogs infected with helminths other than E. granulosus (n = 30), and helminth-free dog sera (n = 20). These findings were compared to those obtained from a somatic extract of protoscoleces (S-Ag). ES-Ag only showed four cross-reacting proteins of 65, 61, 54, and 45-46 kDa. Antigens with apparent masses of 89 and 50 kDa in ES-Ag and of 130 and 67 kDa in S-Ag were identified by sera of dogs infected with E. granulosus only, whereas a protein of 41-43 kDa was recognised by the majority of the sera from dogs with non-echinococcal infection. Employing ELISA to study the same sera, S-Ag revealed higher immunoreactivity than ES-Ag, but also showed higher cross-reactivity levels when sera from dogs with non-echinococcal infection were assayed in immunoblotting.

Shared and non-shared antigens from three different extracts of the metacestode of Echinococcus granulosus

Hydatid cyst fluid (HCF), somatic antigens (S-Ag) and excretory-secretory products (ES-Ag) of Echinococcus granulosus protoscoleces are used as the main antigenic sources for immunodiagnosis of human and dog echinococcosis. In order to determine their non-shared as well as their shared antigenic components, these extracts were studied by ELISA-inhibition and immunoblot-inhibition. Assays were carried out using homologous rabbit polyclonal antisera, human sera from individuals with surgically confirmed hydatidosis, and sera from dogs naturally infected with E. granulosus. High levels of cross-reactivity were observed for all antigenic extracts, but especially for ES-Ag and S-Ag. Canine antibodies evidenced lesser avidity for their specific antigens than antibodies from human origin. The major antigenic components shared by HCF, S-Ag, and ES-Ag have apparent molecular masses of 4-6, 20-24, 52, 80, and 100-104 kDa, including doublets of 41/45, 54/57, and 65/68 kDa. Non-shared polypeptides of each antigenic extract of E. granulosus were identified, having apparent masses of 108 and 78 kDa for HCF, of 124, 94, 83, and 75 kDa for S-Ag, and of 89, 66, 42, 39, 37, and 35 kDa for ES-Ag.

Neutralizing and protective antibodies directed against vaccinia virus envelope antigens.

The infection mechanism of vaccinia virus is largely unknown. Neither the attachment protein of extracellular enveloped virus (EEV), the biologically relevant infectious form of the virus, nor its cellular receptor has been identified. Surprisingly, all former attempts using antibodies to block EEV infection of cells in vitro had failed. Here, we report the production of an anti-envelope hyperimmune serum with EEV neutralizing activity and show that a polyclonal antiserum against the extraviral domain of protein B5R also inhibited EEV infection. In vivo, mice vaccinated with B5R protein were protected against a lethal vaccinia virus challenge. This protectivity is likely to be mediated by neutralizing antibodies. Protein A33R, but not A34R and A36R, also proved to be protective in active and passive vaccination experiments. However, in contrast to B5R, A33R protectivity did not correlate with antibody titers. Because anti-A33R antibodies did not neutralize EEV in vitro, the protectivity mediated by A33R protein probably involves a mechanism different from simple antibody binding. Taken together, our results suggest that antibodies to a specific protective epitope or epitopes on protein B5R are able to prevent EEV infection. The protein encoded by the B5R gene is therefore likely to play a crucial role in the initial steps of vaccinia virus infection-binding to a host cell and entry into its cytoplasm.

Vaccination with a Melan-A peptide selects an oligoclonal T cell population with increased functional avidity and tumor reactivity.

Both the underlying molecular mechanisms and the kinetics of TCR repertoire selection following vaccination against tumor Ags in humans have remained largely unexplored. To gain insight into these questions, we performed a functional and structural longitudinal analysis of the TCR of circulating CD8(+) T cells specific for the HLA-A2-restricted immunodominant epitope from the melanocyte differentiation Ag Melan-A in a melanoma patient who developed a vigorous and sustained Ag-specific T cell response following vaccination with the corresponding synthetic peptide. We observed an increase in functional avidity of Ag recognition and in tumor reactivity in the postimmune Melan-A-specific populations as compared with the preimmune blood sample. Improved Ag recognition correlated with an increase in the t(1/2) of peptide/MHC interaction with the TCR as assessed by kinetic analysis of A2/Melan-A peptide multimer staining decay. Ex vivo analysis of the clonal composition of Melan-A-specific CD8(+) T cells at different time points during vaccination revealed that the response was the result of asynchronous expansion of several distinct T cell clones. Some of these T cell clones were also identified at a metastatic tumor site. Collectively, these data show that tumor peptide-driven immune stimulation leads to the selection of high-avidity T cell clones of increased tumor reactivity that independently evolve within oligoclonal populations.

The promise of advanced imaging techniques for the detection of hepatitis C virus antigens in the infected liver.

Injection drug use before and after liver transplantation: a retrospective multicenter analysis on incidence and outcome. Clin Transplant 2009 DOI: 10.1111/j.1399-0012.2009.01121.x. Background and aims: Injecting drug use (IDU) before and after liver transplantation (LT) is poorly described. The aim of this study was to quantify relapse and survival in this population and to describe the causes of mortality after LT. Methods: Past injection drug users were identified from the LT listing protocols from four centers in Switzerland and France. Data on survival and relapse were collected and used for uni- and multivariate analysis. Results: Between 1988 and 2006, we identified 59 patients with a past history of IDU. The mean age at transplantation was 42.4 yr and the majority of patients were men (84.7%). The indication for LT was for the vast majority viral cirrhosis accounting for 91.5% of cases, while alcoholic cirrhosis was 5.1%. There were 16.9% of patients who had a substitution therapy before and 6.8% who continued after LT. Two patients (3.4%) relapsed into IDU after LT and died at 18 and 41 months. The mean follow-up was 51 months. Overall survival was 84%, 66%, and 61% at 1, 5, and 10 yr after transplantation. Conclusions: Documented IDU was rare in liver transplanted patients. Past IDU was not associated with poorer survival after LT, and relapse after LT occurred in 3.4%.

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2%) and specificities (100% for rMSP5 and 93.8% for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15% (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Cell proliferation and interferon-gamma response to recombinant MBP-3, NarL, MT-10.3, and 16kDa Mycobacterium tuberculosis antigens in Brazilian tuberculosis patients

Human pulmonary tuberculosis (TB) is a worldwide public health problem. In resistant individuals, control of the infection mainly requires development of a Th1 cell immune response with production of cytokines, of which interferon-gamma (IFN-gamma)plays an important role. Several antigens from Mycobacterium tuberculosis complex has been described for use in vaccine development or for diagnostic purposes, however little evaluation has been done in endemic area for TB. The proliferative and IFN-gamma human T cell immune responses, to four recombinant proteins (MBP-3, NarL, MT-10.3, 16 kDa) and PPD, of 38 Brazilian TB patients (6 untreated and 32 treated) and 67 controls (38 positive and 29 negative tuberculin skin test - TST) were compared. The highest reactivity mean rate was obtained with PPD followed by 16 kDa in TB patients. While most of the patients (87%) and controls (> 64%) respond to the PPD, 16kDa was more specifically recognized (> 21%) although less sensitive (54%). When TB patients were divided according to treatment status, opposite to PPD, higher average level of IFN-gamma was induced by 16kDa in untreated (505 pg/ml) compared to treated TB patients and TST+ (269.8 pg/ml x 221.6pg/ml, respectively), although the difference was not significant. These data show that in contrast with the other recombinant proteins, the stimulatory potency of 16kDa to induce proliferative and INF-gamma response was more effective and is more recognized by active TB untreated patients, eliciting in control individuals a more selective immune response than PPD.

Differential lectin labelling of circulating hemocytes from Biomphalaria glabrata and Biomphalaria tenagophila resistant or susceptible to Schistosoma mansoni infection

Lectins/carbohydrate binding can be involved in the Schistosoma mansoni recognition and activation of the Biomphalaria hemocytes. Therefore, expression of lectin ligands on Biomphalaria hemocytes would be associated with snail resistance against S. mansoni infection. To test this hypothesis, circulating hemocytes were isolated from B. glabrata BH (snail strain highy susceptible to S. mansoni), B. tenagophila Cabo Frio (moderate susceptibility), and B. tenagophila Taim (completely resistant strains), labelled with FITC conjugated lectins (ConA, PNA, SBA, and WGA) and analyzed under fluorescence microscopy. The results demonstrated that although lectin-labelled hemocytes were detected in hemolymph of all snail species tested, circulating hemocytes from both strains of B. tenagophila showed a larger number of lectin-labelled cells than B. glabrata. Moreover, most of circulating hemocytes of B. tenagophila were intensively labelled by lectins PNA-FITC and WGA-FITC, while in B. glabrata small hemocytes were labeled mainly by ConA. Upon S. mansoni infection, lectin-labelled hemocytes almost disappeared from the hemolymph of Taim and accumulated in B. glabrata BH. The role of lectins/carbohydrate binding in resistance of B. tengophila infection to S. mansoni is still not fully understood, but the data suggest that there may be a correlation to its presence with susceptibility or resistance to the parasite.