TLR5-dependent immunogenicity of a recombinant fusion protein containing an immunodominant epitope of malarial circumsporozoite protein and the FliC flagellin of Salmonella Typhimurium

Recently, we described the improved immunogenicity of new malaria vaccine candidates based on the expression of fusion proteins containing immunodominant epitopes of merozoites and Salmonella enterica serovar Typhimurium flagellin (FliC) protein as an innate immune agonist. Here, we tested whether a similar strategy, based on an immunodominant B-cell epitope from malaria sporozoites, could also generate immunogenic fusion polypeptides. A recombinant His6-tagged FliC protein containing the C-terminal repeat regions of the VK210 variant of Plasmodium vivax circumsporozoite (CS) protein was constructed. This recombinant protein was successfully expressed in Escherichia coli as soluble protein and was purified by affinity to Ni-agarose beads followed by ion exchange chromatography. A monoclonal antibody specific for the CS protein of P. vivax sporozoites (VK210) was able to recognise the purified protein. C57BL/6 mice subcutaneously immunised with the recombinant fusion protein in the absence of any conventional adjuvant developed protein-specific systemic antibody responses. However, in mice genetically deficient in expression of TLR5, this immune response was extremely low. These results extend our previous observations concerning the immunogenicity of these recombinant fusion proteins and provide evidence that the main mechanism responsible for this immune activation involves interactions with TLR5, which has not previously been demonstrated for any recombinant FliC fusion protein.

Use of an immunodominant p17 antigenic fraction of Neospora caninum in detection of antibody response in cattle

A Neospora caninum 17 kDa protein fraction (p17) has been described as an immunodominant antigen (IDA) under reducing and non-reducing conditions. The aim of the present study was to investigate the diagnostic utility of p17 in cattle. In order to achieve this, p17 was purified by electroelution from whole N. caninum tachyzoite soluble extract and a p17-based Western blot (WB-p17) was developed. The p17 recognition was measured by densitometry and expressed as OD values to check the validity of the WB-p17. A total of 131 sera including sequential samples from naturally- and experimentally-infected calves and breeding cattle were analysed by WB-p17 and compared with IFAT using whole formalin-fixed tachyzoites as a reference test. The results obtained highlight the feasibility of using the N. caninum p17 in a diagnostic test in cattle. Firstly, the assay based on the p-17 antigen discriminated between known positive and negative sera from different cattle populations, breeding cattle and calves. Secondly, the p17 antigen detected fluctuations in the antibody levels and seroconversion in naturally- and experimentally-infected cattle. Significant differences in p-17 antigen recognition were observed between naturally infected aborting and non-aborting cattle, as well as significant antibody fluctuations over time in experimentally infected cattle, which varied between groups. Furthermore, the results obtained with WB-p17 are in accordance with the results obtained with the IFAT, as high agreement values were obtained when all bovine subpopulations were included (kappa = 0.86).

The non-palindromic adaptor-PCR method for the identification of the T-cell receptor genes of an interferon-gamma-secreting T-cell hybridomaspecific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen

Cloning of the T-cell receptor genes is a critical step when generating T-cell receptor transgenic mice. Because T-cell receptor molecules are clonotypical, isolation of their genes requires reverse transcriptase-assisted PCR using primers specific for each different Valpha or Vß genes or by the screening of cDNA libraries generated from RNA obtained from each individual T-cell clone. Although feasible, these approaches are laborious and costly. The aim of the present study was to test the application of the non-palindromic adaptor-PCR method as an alternative to isolate the genes encoding the T-cell receptor of an antigen-specific T-cell hybridoma. For this purpose, we established hybridomas specific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen. These T-cell hybridomas were characterized with regard to their ability to secrete interferon-gamma, IL-4, and IL-10 after stimulation with the antigen. A CD3+, CD4+, CD8- interferon-gamma-producing hybridoma was selected for the identification of the variable regions of the T-cell receptor by the non-palindromic adaptor-PCR method. Using this methodology, we were able to rapidly and efficiently determine the variable regions of both T-cell receptor chains. The results obtained by the non-palindromic adaptor-PCR method were confirmed by the isolation and sequencing of the complete cDNA genes and by the recognition with a specific antibody against the T-cell receptor variable ß chain. We conclude that the non-palindromic adaptor-PCR method can be a valuable tool for the identification of the T-cell receptor transcripts of T-cell hybridomas and may facilitate the generation of T-cell receptor transgenic mice.

Análise proteômica das principais proteínas antigênicas do veneno da vespa agelaia pallipes / Proteomic analysis of mayor antigenic proteins from agelaia pallipes venom

Objetivo: alguns pacientes alérgicos ao veneno de vespas apresentam pesquisa negativa de IgE específica com os extratos disponíveis. Para investigar esta falta de reação cruzada, este estudo pretende caracterizar os antígenos principais do veneno de uma das espécies encontradas no Brasil, a Agelaia pallipes, utilizando a análise proteômica. Método: realizamos eletroforese bidimensional com veneno de Agelaia pallipes. Na primeira dimensão utilizamos tiras de gel de 7 cm com gradiente de pH de 3.0 -10.0, e na segunda SDS-PAGE 15%. Com géis feitos em duplicata, o primeiro foi transferido para nitrocelulose e incubado com o soro de paciente sensibilizado (diluição 1:5). A imunodetecção foi realizada com anti-IgE humana biotinilada e ECL (Enhanced Chemiluminescence). No segundo gel, corado Coomassie, os spots correspondentes às proteínas reconhecidas pela IgE através do immuniblotting foram processados e analisados no espectrometro de massa do tipo MALDI-ToF. A identificação foi obtida por PMF - Peptide Mass Fingerprinting. Resultados: a eletroforese bidimensional com o veneno da Agelaia pallipes evidenciou várias proteínas com peso molecular (PM) abaixo de 20kDa. Com immunoblotting foram detectadas proteínas reconhecidas pela IgE com PM entre 20 e 38 kDa. Pela análise proteômica, estas proteínas foram identificadas principalmente como antígeno 5 e serino-proteases. Conclusão: este é o primeiro trabalho a identificar alérgenos de vespas neotropicais com análise proteômica. Além do antígeno 5, identificamos serino-proteases que apenas recentemente foram citadas neste tipo de amostra biológica, mostrando semelhança parcial entre estas proteínas de venenos de vertebrados (serpentes). Nosso projeto futuro será o sequenciamento das amostras.

Antibody recognition of synthetic peptides mimicking immunodominant regions of HIV-1 p24 and p17 proteins / Reconocimiento por anticuerpos de péptidos sintéticos que imitan regiones inmunodominantes de las proteínas p24 y p17 de VIH-1

The gag gene of HIV-1 encodes a single open reading frame of 55 kDa that contains three subdomains: the matrix domain (p17), the capsid domain (p24) and the nucleocapsid domain (p15). The p24 and p17 proteins have a predominant a-helical structure and perform important functions throughout thevirallife-cycle. The determination of gag-specific antibodies is important because declining titers of these antibodies herald clinical deterioration.In this work we present the results obtained on immunoreactiviy of synthetic peptides that mimic immunogenic a-helical regions of p24 and p17. The influence on the immunoreactivity of structural modifications in native sequences, including the addition of non immunogenic side chains: AAAC- and -CAAA on both side of minimal epitopes was evaluated in indirect and competitive enzymeimmunoassays. The conformational characteristcs to the peptides were analysed by circular dichroism and these results were correlated with that obtained in the immunoassays. It was shown that the reactivity of peptides mimicking short a-helical regions of p24 and p17 is improved by adding short non immunogenic chains on both N- and C- terminus. These modifications enhanced the immobilization of the peptides onto the solid support and allowed more accesibility to the minimal epitopes byspecific antibodies, in solution.

El gen gag del VIH-1 codifica una región de 55kDA que contiene tres subdominios: matriz (p17), cápside (p24) y nucleocápside (p15). Las proteínas p24 y p17 tienen una estructura predominante helicoidal y cumplen un rol importante en el ciclo de vida del virus. En este trabajo presentamos los resultados de inmunorreactividad de péptidos sintéticos que imitan regiones helicoidales de p24 y p17. Utilizando enzimoinmunoensayos se evaluó la influencia de modificaciones en las secuencias nativas sobre la capacidad de reconocimiento de anticuerpos específicos en solución y en fase sólida, incluyendo el agregado de cadenas no inmunogénicas en ambos extremos de los epitopes mínimos. La conformación de los péptidos se determinó por dicroísmo circular y los resultados se correlacionaron con los de inmunorreactividad. Se observó que la capacidad de reconocimiento de anticuerpos por péptidos pequeños que imitan estructuras helicoidales de p24 y p17 mejoró con el agregado de cadenas no inmunogénicas en ambos extremos de los epitopes. Estas modificaciones mejoran la inmovilización sobre las superficies sólidas y permiten una mayor accesibilidad de los anticuerpos a los epitopes mínimos en solución.

Vaccine development against Theileria parasite.

Bovine piroplasmosis caused by Theileria sergenti is a major cause of economical loss in grazing cattle in Japan. We found that parasite stocks and isolates consist of genetically and antigenically mixed population. To differentiate parasite populations bearing 2 allelic forms of p32, an immunodominant piroplasm surface protein, 2 sets of oligonucleotide primers were designed to amplify either of the 2 alleles by polymerase chain reaction (PCR). By using this allele-specific PCR, we found that the majority of T. sergenti-infected calves in Japan harbored mixed parasite populations with C and I type parasites. Amino acid sequence of p32 contains Lys-Glu-Lys (KEK) motif which is one of tripeptide necessary for malaria parasite to invade erythrocytes. We produced 2 vaccine candidates, recombinant baculovirus p32 and synthetic peptide containing KEK motif. Immunization of either recombinant p32 or synthetic peptide containing a KEK sequence with adjuvant resulted in low parasitemia and reduced the clinical symptoms compared to control calves. Interestingly, parasites with a p32 allelic form corresponding to one used as the immunogen were suppressed. Therefore, a cocktail vaccine containing KEK peptides derived from C and I type parasites is desired for control Theileria parasite infection in Japan.

Cellular immune response to retinal S-antigen & interphotoreceptor retinoid binding protein fragments in idiopathic human uveitis.

The role of retinal antigens in idiopathic human uveitis has been studied in 38 patients of uveitis, and 30 patients of systemic connective tissue disease (CTD) and 30 healthy volunteers. Lymphocyte proliferative responses were tested in vitro against native S-antigen, its uveitopathogenic peptides (peptide M, peptide G), yeast histone H3 peptide and uveitopathogenic fragment of interphotoreceptor retinoid binding protein (IRBP: R16) to establish their role in pathogenesis of human uveitis. Seven patients with uveitis, and none among CTD patients and healthy volunteers, responded (stimulation index > 3) to at least one retinal antigen used. One uveitis patient showed response to native S-antigen, peptide M and yeast histone H3. One responded to both S-antigen and peptide M and another responded to both peptide G and R16 peptide. Two responded to S-antigen only, one to peptide M and one to peptide G. In addition, one uveitis patient responded to yeast histone H3 only. These results suggest that retinal antigens may play a role in the etiopathogenesis of a subset of idiopathic human uveitis.