Vaccination With Recombinant Filamentous fd Phages Against Parasite Infection Requires TLR9 Expression.

Recombinant filamentous fd bacteriophages (rfd) expressing antigenic peptides were shown to induce cell-mediated immune responses in the absence of added adjuvant, being a promising delivery system for vaccination. Here, we tested the capacity of rfd phages to protect against infection with the human protozoan Trypanosoma cruzi, the etiologic agent of Chagas Disease. For this, C57BL/6 (B6) and Tlr9-/- mice were vaccinated with rfd phages expressing the OVA257-264 peptide or the T. cruzi-immunodominant peptides PA8 and TSKB20 and challenged with either the T. cruzi Y-OVA or Y-strain, respectively. We found that vaccination with rfd phages induces anti-PA8 and anti-TSKB20 IgG production, expansion of Ag-specific IFN-γ, TNF-α, and Granzyme B-producing CD8+ T cells, as well as in vivo Ag-specific cytotoxic responses. Moreover, the fd-TSKB20 vaccine was able to protect against mortality induced by a high-dose inoculum of the parasite. Although vaccination with rfd phages successfully reduced both parasitemia and parasite load in the myocardium of WT B6 mice, Tlr9-/- animals were not protected against infection. Thus, our data extend previous studies, demonstrating that rfd phages induce Ag-specific IgG and CD8+ T cell-mediated responses and confer protection against an important human parasite infection, through a TLR9-dependent mechanism.

Development of a highly sensitive noncompetitive electrochemical immunosensor for the detection of atrazine by phage anti-immunocomplex assay.

The development of immunosensors for the detection of small molecules is of great interest because of their simplicity, high sensitivity and extended analytical range. Due to their size, small compounds cannot be simultaneously recognized by two antibodies impeding their detection by noncompetitive two-site immunoassays, which are superior to competitive ones in terms of sensitivity, kinetics, and working range. In this work, we combine the advantages of magneto-electrochemical immunosensors with the improved sensitivity and direct proportional signal of noncompetitive immunoassays to develop a new Phage Anti-Immunocomplex Electrochemical Immunosensor (PhAIEI) for the detection of the herbicide atrazine. The noncompetitive assay is based on the use of recombinant M13 phage particles bearing a peptide that specifically recognizes the immunocomplex of atrazine with an anti-atrazine monoclonal antibody. The PhAIEI performed with a limit of detection (LOD) of 0.2 pg mL(-1), which is 200-fold better than the LOD obtained using the same antibody in an optimized conventional competitive ELISA, with a large increase in working range. The developed PhAIEI was successfully used to assay undiluted river water samples with no pretreatment and excellent recoveries. Apart from the first demonstration of the benefits of integrating phage anti-immunocomplex particles into electrochemical immunosensors, the extremely low and environmentally relevant detection limits of atrazine attained with the PhAIEIS may have direct applicability to fast and sensitive detection of this herbicide in the environment.

Novel amyloid-beta specific scFv and VH antibody fragments from human and mouse phage display antibody libraries.

Anti-amyloid immunotherapy has been proposed as an appropriate therapeutic approach for Alzheimer's disease (AD). Significant efforts have been made towards the generation and assessment of antibody-based reagents capable of preventing and clearing amyloid aggregates as well as preventing their synaptotoxic effects. In this study, we selected a novel set of human anti-amyloid-beta peptide 1-42 (Abeta1-42) recombinant monoclonal antibodies in a single chain fragment variable (scFv) and a single-domain (VH) format. We demonstrated that these antibody fragments recognize in a specific manner amyloid-beta deposits in APP/Tg mouse brains, inhibit toxicity of oligomeric Abeta1-42 in neuroblastoma cell cultures in a concentration-dependent manner and reduced amyloid deposits in APP/Tg2576 mice after intracranial administration. These antibody fragments recognize epitopes in the middle/C-terminus region of Abeta, which makes them strong therapeutic candidates due to the fact that most of the Abeta species found in the brains of AD patients display extensive N-terminus truncations/modifications.

Inexpensive anti-cysticercosis vaccine: S3Pvac expressed in heat inactivated M13 filamentous phage proves effective against naturally acquired Taenia solium porcine cysticercosis.

In search of reducing vaccine production costs', a recombinant M13 phage version of the anti-cysticercosis tripeptide vaccine (S3Pvac) was developed. The efficacy of S3Pvac-Phage vs. placebo was evaluated in a randomized trial that included 1,047 rural pigs in 16 villages of Central Mexico. Three to five months after vaccination 530 pigs were examined by tongue inspection. At 5-27 months of age, 331 pigs (197 vaccinated/134 controls) were inspected at necropsy. Vaccination reduced 70% the frequency of tongue cysticercosis and, based on necropsy, 54% of muscle-cysticercosis and by 87% the number of cysticerci.

Detection of constitutive molecules on Histoplasma capsulatum yeasts through single chain variable antibody fragments displayed in M13 phages.

A nonimmune library, containing single chain variable fragments (scFv) of immunoglobulin human genes displayed on the surface of M13 filamentous phages, was used to recognize molecules exposed on Histoplasma capsulatum yeasts' surface, during their growth in synthetic medium. The scFv clones were checked in their consistency by Dot-ELISA using HRP/anti-M13 conjugate, and they were tested to recognize molecules on H. Capsulatum yeasts' surface by ELISA in plates. Three out of 80 scFv cones (C2, C6, and C52) reacted consistently with H. capsulatum molecules, and they recognized molecules from both H. capsulatum morphologic phases. However, C6 and C52 clones reacted better with molecules on the surface of whole yeasts, with molecules from the yeasts' cell-wall extract, and with molecules released to the supernatant of the yeast culture. Mycelial supernatants from other fungi, as well as from a Mycobacterium filtrate, were not recognized by scFv phage monoclones. Monoclones C2, C6, and C52 recognized yeast molecules irrespective of the H. capsulatum strains used; the C6 clone revealed a specific immunohistochemistry reaction when tested against homologous and heterologous fungal infected tissues. The scFv clones isolated will be a useful toll to define the role of their target molecules in the host-parasite relationship of histoplasmosis.

Recombinant bacteriophage-based multiepitope vaccine against Taenia solium pig cysticercosis.

The aim of this study was to test the capacity of recombinant phages to deliver antigens for vaccination against porcine cysticercosis. Thus, three peptides (KETc1, KETc12, GK1) and a recombinant antigen KETc7, previously proven to induce high levels of protection against pig cysticercosis, were expressed on the surface of the M13 bacteriophage at multiple copies. The pool of these four recombinant phages induced high levels of protection against an experimental murine cysticercosis. The immunogenicity of the phage vaccine preparation was therefore, tested in pigs, the natural host of Taenia solium. Subcutaneous or oral vaccination with these phages induced antigen-specific cellular immune responses in pigs. Preliminary data also points to the protective capacity of this recombinant phage vaccine against pig cysticercosis. The immunogenicity of these recombinant phages, together with the low cost of their production, make them a realistic candidate to be tested in pigs as an anti-cysticercus phage vaccine for field trials. This is the first report describing the application of a filamentous bacteriophage as a vaccine in large animals such as pigs, the only intermediate hosts of T. solium, a parasite of major medical importance in developing countries. The potential application of phages as a modern platform for vaccines for human and animal diseases is discussed.

Amyloid-beta peptide-specific single chain Fv antibodies isolated from an immune phage display library.

A single-chain fragment variable (scFv) antibody library displayed on phage was constructed using spleen cells from mice immunized with human amyloid-beta peptide (Abeta42). This first anti-Abeta42 scFv immune antibody library was selected against human Abeta42. A number of positive clones were obtained, and sequences of VH and Vkappa genes were analyzed using ExPASy and BLAST computer tools. This analysis revealed that only two unique clones with identical VH and Vkappa complementarity determining region (CDR) (except HCDR2) and identical germline genes were selected, indicating that oligoclonal immune response was occurring in Abeta42-immunized mice. Abeta42-specific scFv antibodies selected from this first immune anti-Abeta42 phage antibody library may be an important tool for the development of therapeutic molecules for Alzheimer's disease (AD).

Immunisation with phage-displayed variable region 2 from meningococcal PorA outer membrane protein induces bactericidal antibodies against Neisseria meningitidis.

The immunogenicity and functional activity of antibodies raised in mice against the cyclic disulphide peptide corresponding to the variable region 2 of PorA outer membrane protein from Neisseria meningitidis strain B385 (serosubtype P1.15), displayed on filamentous phage, were evaluated. The epitope, flanked either by cysteine or cysteine and three glycine residues, was expressed as a fusion to PVIII protein from M13. Immunisation of Balb/C mice with either phage generated antibody specific responses. Sera raised against the phage exposing the cyclic peptide through the three-glycine linker recognised the native protein better than those raised against the peptide with no linker. Only the phage displaying the cyclic peptide with linker was capable of inducing antibodies with bactericidal activity. These results indicate the possibility of using phage display for conformational peptide expression for immunisation to elicit functional antibody responses.

Identification of mimotopes of platelet autoantigens associated with autoimmune thrombocytopenic purpura.

GPIIb/IIIa, the human platelet glycoprotein complex, is the autoantigen most commonly recognized by autoantibodies in autoimmune thrombocytopenic purpura (AITP). Two murine monoclonal antibodies (mAbs), namely Y2/51 and 5B12, directed against gpIIIa and gpIIb/IIIa, respectively, and rabbit anti-human platelet polyclonal antibodies have been used to select AITP-related epitopes from a phage display peptide library expressing random dodecapeptides in the pIII coat protein of M13 phage. The selected phage clones were tested by ELISA for binding to rabbit anti-human platelet antibodies as well as to sera from AITP patients. Seven clones reacted strongly with rabbit anti-human platelet antibodies, and four clones reacted with sera from AITP patients. Some homology between peptide inserts sequences of selected clones and human platelet gpIIIa and gpIb were found.

Monoclonal antibodies against the major coat protein of filamentous phage as a useful analytical tool for bacteriophage peptide/protein display.

Four mouse monoclonal antibodies (MAbs) that react with filamentous M13KO7 and R408 phage were obtained. Three of these MAbs (two IgG2a and one IgG3) recognize linear sequences of the p8 main structural coat protein, and one (IgG2a) identifies a putatively conformational epitope, as suggested by Western blot. These MAbs also react with recombinant phage expressing peptide antigens fused to p8, and are though useful reagents for peptide/protein phage display screening based methods. The latter was shown in an enzyme-linked immunoadsorbent assay (ELISA) and a visual immunoassay where one of the anti-p8 MAbs was used to capture recombinant phages displaying a peptide characteristic of the Hepatitis B virus surface antigen or a Dengue virus-related peptide antigen.