Immunogenicity, antibody responses and vaccine efficacy of recombinant annexin B30 against Schistosoma mansoni.

AIMS: Schistosomes infect approximately 250 million people worldwide. To date, there is no effective vaccine available for the prevention of schistosome infection in endemic regions. There remains a need to develop means to confer long-term protection of individuals against reinfection. In this study, an annexin, namely annexin B30, which is highly expressed in the tegument of Schistosoma mansoni was selected to evaluate its immunogenicity and protective efficacy in a mouse model. METHODS AND RESULTS: Bioinformatics analysis showed that there were three potential linear B-cell epitopes and four conformational B-cell epitopes predicted from annexin B30, respectively. Full-length annexin B30 was cloned and expressed in Escherichia coli BL21(DE3). In the presence of adjuvants, the soluble recombinant protein was evaluated for its protective efficacy in two independent vaccine trials. Immunization of CBA mice with recombinant annexin B30 formulated either in alum only or alum/CpG induced a mixed Th1/Th2 cytokine profile but no significant protection against schistosome infection was detected. CONCLUSION: Recombinant annexin B30 did not confer significant protection against the parasite. The molecule may not be suitable for vaccine development. However, it could be an ideal biomarker recommended for immunodiagnostics development.

Expression of the Tpanxb1 gene from Taenia pisiformis and its potential diagnostic value by dot-ELISA.

Cysticercosis, caused by the larvae of Taenia pisiformis, is a common disease in rabbits that results in economic losses. To date, there has been limited information available on the early detection of infection by this parasite. This study describes a dot-ELISA method based on an autologous antigen annexin B1 (Tpanxb1). Its potential for serodiagnosis of rabbit cysticercosis was also evaluated. Western blot analysis revealed that the recombinant Tpanxb1 (rTpanxb1) protein could be specifically recognized by rabbit anti-sera. In serum trials, the antibodies could be detected by dot-ELISA using rTpanxb1 at 14 days post-infection. The positive response was present for up to 49 days post-infection. Based on the necropsy results of 169 rabbit samples, the relative sensitivity and specificity of the dot-ELISA were 94.55% and 92.86%, respectively. This study provides a foundation for studying the immunological function of annexin and its application to control Taenia cestodes.

Identification of annexin 1 as a novel autoantigen in acute exacerbation of idiopathic pulmonary fibrosis.

Consistent with the hypothesis that pulmonary epithelial apoptosis is the key to the acute exacerbation of idiopathic pulmonary fibrosis (IPF), we conducted serological identification of Ags by recombinant expression cloning (SEREX) analysis using type II alveolar cell carcinoma (A549) cell lines to identify disease-related Abs. In a survey of Abs to the recombinant autoantigens identified by SEREX analysis, five Abs were identified as novel candidates for the acute exacerbation of IPF. Abs to annexin 1 were detected in 47 and 53% of the sera and bronchoalveolar lavage materials from patients with acute exacerbation of IPF. Some identical TCR Vbeta genes were identified in sequential materials obtained at 1-3 mo in all 10 acute exacerbation IPF cases, suggesting that some infiltrating CD4-positive T cells sharing limited epitopes expand by Ag-driven stimulation during disease extension. The CDR3 region of these identical TCR Vbeta genes showed high homology with the N-terminal portion of annexin 1, including in the HLA-DR ligand epitopes predicted by TEPITOPE analysis. By Western blotting analysis and observation of the CD4-positive T cell responses in bronchoalveolar lavage samples, the N-terminal portion of annexin 1 was cleaved and found to induce marked proliferative responses of CD4-positive T cells in three patients. Our study demonstrates that annexin 1 is an autoantigen that raises both Ab production and T cell response in patients with acute exacerbation of IPF, and that the N-terminal portion of annexin 1 plays some role in the pathogenesis of acute exacerbation in IPF patients.

Pemphigus vulgaris antibody identifies pemphaxin. A novel keratinocyte annexin-like molecule binding acetylcholine.

Because pemphigus vulgaris (PV) IgGs adsorbed on the rDsg3-Ig-His baculoprotein induced blisters in neonatal mice, it was proposed that anti-desmoglein 3 (Dsg 3) autoantibody causes PV. However, we found that rDsg3-Ig-His absorbs autoantibodies to different antigens, including a non-Dsg 3 keratinocyte protein of 130 kDa. This prompted our search for novel targets of PV autoimmunity. The PV IgG eluted from a 75-kDa keratinocyte protein band both stained epidermis in a pemphigus-like pattern and induced acantholysis in keratinocyte monolayers. Screening of a keratinocyte lambdagt11 cDNA library with this antibody identified clones carrying cDNA inserts encoding a novel molecule exhibiting approximately 40% similarity with annexin-2, named pemphaxin (PX). Recombinant PX (rPX-His) was produced in Escherichia coli M15 cells, and, because annexins can act as cholinergic receptors, its conformation was tested in a cholinergic radioligand binding assay. rPX-His specifically bound [(3)H]acetylcholine, suggesting that PX is one of the keratinocyte cholinergic receptors known to be targeted by disease-causing PV antibodies. Preabsorption of PV sera with rPX-His eliminated acantholytic activity, and eluted antibody immunoprecipitated native PX. This antibody alone did not cause skin blisters in vivo, but its addition to the preabsorbed PV IgG fraction restored acantholytic activity, indicating that acantholysis in PV results from synergistic action of antibodies to different keratinocyte self-antigens, including both acetylcholine receptors and desmosomal cadherins.

Protein kinase C and casein kinase 2 phosphorylate in vitro proteins of the annexin family from eggs of loach Misgurnus fossilis.

A mixture of proteins of the annexin family was obtained from the cytoplasm of mature eggs of loach Misgurnus fossilis (by reprecipitation with acid phospholipids in the presence of Ca2+). This mixture comprised five proteins with molecular weights of 58, 38, 36, 35, and 31 kD. Polyclonal rabbit antibodies against the major 31-kD protein were obtained. Western blot analysis showed that the obtained antibodies exhibit a high specificity towards the 31-kD protein from eggs and other tissues of loach and zebrafish (Brachydanio rerio). The analysis of cDNA corresponding to the 31-kD protein by screening the zebrafish cDNA library confirmed that this protein belongs to the annexin family. Phosphorylation of the obtained annexins in vitro was studied. It is shown that the 58-kD protein is phosphorylated by casein kinase 2 (CK2), whereas the 38-, 36-, 35-, and 31-kD proteins are phosphorylated by protein kinase C (PKC).

Immunoreactivities of p11 and calcyclin in baker's yeast, wheat germ and lobster.

After effectively eliminating the nonspecific cross-immunoreactivity with the affinity columns of anti-IgG agarose and IgG agarose, the potent immunoreactivities of p11 and calcyclin in wheat germ, lobster tail muscle, and three strains of baker's yeast were analyzed by Western blotting using mouse anti-p11 and rabbit anti-calcyclin. The occurrence of multiple bands may be due to either autolyses and/or the interactions between the p11 (or calcyclin) and other endogenous biological molecules. The results suggest not only a ubiquitous distribution and a universal Ca(2+)-mediating regulatory role of p11 and calcyclin in eukaryotes, but also an evolutionary conservation of these (S-100)-related proteins.

Annexin XIIIb: a novel epithelial specific annexin is implicated in vesicular traffic to the apical plasma membrane.

The sorting of apical and basolateral proteins into vesicular carriers takes place in the trans-Golgi network (TGN) in MDCK cells. We have previously analyzed the protein composition of immunoisolated apical and basolateral transport vesicles and have now identified a component that is highly enriched in apical vesicles. Isolation of the encoding cDNA revealed that this protein, annexin XIIIb, is a new isoform of the epithelial specific annexin XIII sub-family which includes the previously described intestine-specific annexin (annexin XIIIa; Wice, B. M., and J. I. Gordon. 1992. J. Cell Biol. 116:405-422). Annexin XIIIb differs from annexin XIIIa in that it contains a unique insert of 41 amino acids in the NH2 terminus and is exclusively expressed in dog intestine and kidney. Immunofluorescence microscopy demonstrated that annexin XIIIb was localized to the apical plasma membrane and underlying punctate structures. Since annexins have been suggested to play a role in membrane-membrane interactions in exocytosis and endocytosis, we investigated whether annexin XIIIb is involved in delivery to the apical cell surface. To this aim we used permeabilized MDCK cells and a cytosol-dependent in vitro transport assay. Antibodies specific for annexin XIIIb significantly inhibited the transport of influenza virus hemagglutinin from the TGN to the apical plasma membrane while the transport of vesicular stomatitis virus glycoprotein to the basolateral cell surface was unaffected. We propose that annexin XIIIb plays a role in vesicular transport to the apical plasma membrane in MDCK cells.

The 56K autoantigen is identical to human annexin XI.

Anti-56K autoantibodies are present in sera from patients with various autoimmune diseases, predominantly in sera from patients with rheumatoid arthritis, systemic lupus erythematosus, or Sjögren's syndrome. To clarify the molecular structure of this autoantigen, we isolated a 2.0-kilobase pair cDNA clone considered to encode the full-length 56K autoantigen. The longest open reading frame encodes a 505-amino acid polypeptide, with a predicted molecular mass of 54.4 kDa. The in vitro translated protein is recognized by all anti-56K positive patient sera tested. Antibodies affinity-purified using the bacterially expressed recombinant protein recognized the 56K autoantigen in a HeLa cell extract. cDNA sequencing revealed that the 56K cDNA shares a high degree of homology in both nucleotide (87%) and amino acid sequence (92.5%) with bovine annexin XI, indicating that the 56K cDNA encodes the human homologue of annexin XI, a member of the Ca(2+)-dependent phospholipid binding protein family. Anti-56K autoantibody exhibits both a cytoplasmic and a nuclear staining in immunofluorescence experiments. Patients' sera recognize preferentially the N-terminal region of the protein, which is specific for 56K/annexin XI and not shared by other annexins, indicating that the autoimmune response to 56K/annexin XI in these patients is specific for this annexin family member.