Defining a protective epitope on factor H binding protein, a key meningococcal virulence factor and vaccine antigen.

Mapping of epitopes recognized by functional monoclonal antibodies (mAbs) is essential for understanding the nature of immune responses and designing improved vaccines, therapeutics, and diagnostics. In recent years, identification of B-cell epitopes targeted by neutralizing antibodies has facilitated the design of peptide-based vaccines against highly variable pathogens like HIV, respiratory syncytial virus, and Helicobacter pylori; however, none of these products has yet progressed into clinical stages. Linear epitopes identified by conventional mapping techniques only partially reflect the immunogenic properties of the epitope in its natural conformation, thus limiting the success of this approach. To investigate antigen-antibody interactions and assess the potential of the most common epitope mapping techniques, we generated a series of mAbs against factor H binding protein (fHbp), a key virulence factor and vaccine antigen of Neisseria meningitidis. The interaction of fHbp with the bactericidal mAb 12C1 was studied by various epitope mapping methods. Although a 12-residue epitope in the C terminus of fHbp was identified by both Peptide Scanning and Phage Display Library screening, other approaches, such as hydrogen/deuterium exchange mass spectrometry (MS) and X-ray crystallography, showed that mAb 12C1 occupies an area of ∼1,000 Å(2) on fHbp, including >20 fHbp residues distributed on both N- and C-terminal domains. Collectively, these data show that linear epitope mapping techniques provide useful but incomplete descriptions of B-cell epitopes, indicating that increased efforts to fully characterize antigen-antibody interfaces are required to understand and design effective immunogens.

The Spr1875 protein confers resistance to the microglia-mediated killing of Streptococcus pneumoniae.

By screening a whole-genome λ-display library of Streptococcus pneumoniae, we have previously identified a novel surface protein, named Spr1875, that exhibited immunogenic properties and was closely related to pneumococcal virulence. In the present study, we investigated the role of the Spr1875 antigen in the interaction of S. pneumoniae with microglia, the resident brain macrophages. By using an in vitro infection model, the BV2 microglial cell line was challenged with the S. pneumoniae strain DP1004 and its isogenic spr1875-deleted mutant (Δspr1875). Both strains were phagocytosed by microglia efficiently and to a similar extent; however, the DP1004 strain was more resistant than the Δspr1875 mutant to the intracellular killing, as assessed by antibiotic protection and phagosome maturation assays. Moreover, significant differences between the two strains were also observed in terms of susceptibility to microglia-mediated killing. Taken together, these results indicate that S. pneumoniae-microglial cell interplay is influenced by the presence of Spr1875, suggesting that this protein may play a role in the pathogenesis of pneumococcal meningitis.

Novel immunogenic peptides elicit systemic anaphylaxis in mice: implications for peptide vaccines.

Peptide-based therapies are showing increasing potential for the development of vaccines and in the treatment of many important diseases. We previously reported two peptide conjugate vaccines that protected mice against pneumococcal disease. During this study, we observed an unexpected phenomenon; several vaccine candidates induced a rapid, fatal anaphylaxis after booster injection of the peptide conjugate. Further investigation indicated the reaction was mediated by the production of peptide-specific IgE and the release of histamine. Notably, among seven peptides tested, all of which bound the same mAb that selected them from a phage library, only four elicited this severe reaction. Sequence alignment analysis of all peptides revealed unique clusters of acidic amino acid residues in the allergenic peptides. Substitution of the acidic amino acid residues, ED, of peptide MP2 with their amine equivalents, QN, eliminated the anaphylactic effects but did not affect the production of peptide-specific IgG. These results have important implications for both the study of allergens and the development of future peptide-based therapies.

Plasminogen- and fibronectin-binding protein B is involved in the adherence of Streptococcus pneumoniae to human epithelial cells.

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. The ability of this bacterium to adhere to epithelial cells is considered as an essential early step in colonization and infection. By screening a whole genome phage display library with sera from infected patients, we previously identified three antigenic fragments matching open reading frame spr0075 of the strain R6 genome. This locus encodes for an approximately 120-kDa protein, herein referred to as plasminogen- and fibronectin-binding protein B (PfbB), which displays an LPXTG cell wall anchoring motif and six repetitive domains. In this study, by using isogenic pfbB-deleted mutants of the encapsulated D39 and of the unencapsulated DP1004 type 2 pneumococcal strains, we show that PfbB is involved in S. pneumoniae adherence to various epithelial respiratory tract cell lines. Our data suggest that PfbB directly mediates bacterial adhesion, because fluorescent beads coated with the recombinant PfbB sp17 fragment (encompassing one of the six repetitive domains and the C-terminal region) efficiently bound to epithelial cells. Mutants lacking PfbB bound to fibronectin and plasminogen considerably less efficiently than wild type bacteria, whereas sp17-coated beads specifically bound to both of these substrates. Taken together, our data suggest that, by directly interacting with fibronectin, PfbB significantly increases the ability of S. pneumoniae to adhere to human epithelial cells.

Peptide mimics of two pneumococcal capsular polysaccharide serotypes (6B and 9V) protect mice from a lethal challenge with Streptococcus pneumoniae.

Anti-polysaccharide immunity is a key facet of protection against several bacterial pathogens. Problems exist with current polysaccharide vaccines and alternative strategies that deliver a protective response are needed. We have identified immunological peptide mimics of type 6B and 9V pneumococcal capsular polysaccharides that could be used as vaccine antigens. Peptides mimicking antigenic properties of serotype 6B capsular polysaccharide were obtained from a phage-displayed peptide library expressing dodecameric peptides, using a human monoclonal antibody (Db3G9). A murine monoclonal antibody (206, F-5) against the serotype 9V capsular polysaccharide identified three peptide mimotopes from the dodecameric peptide library and one from a random pentadecameric peptide library. In ELISA, binding of 206, F-5 and Db3G9 to phage displaying the selected mimotopes was significantly inhibited by type-specific pneumococcal polysaccharide. Peptides were conjugated to keyhole limpet haemocyanin and were used to immunise mice. Two peptides, MP13 and MP7, induced specific anti-6B and 9V polysaccharide antibodies, respectively. Mice immunised with MP7-keyhole limpet hemocyanin or MP13-keyhole limpet hemocyanin conjugate were significantly and specifically protected against a lethal challenge with pneumococci of the appropriate serotype. This study provides strong in vivo evidence that peptide mimics are alternatives to polysaccharide vaccines.

Specific and selective probes for Pseudomonas aeruginosa from phage-displayed random peptide libraries.

The design of novel biosensors for the detection of biological threats, such as Pseudomonas aeruginosa, requires probes that specifically bind biological agents and insure their immediate and efficient recognition. Advanced bio-selective sensors may meet the requests for isolation, concentration of the agents and their real-time detection. There is a need for robust and inexpensive affinity probes alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we identified from two phage-displayed random peptide libraries phage clones displaying peptides capable of specific and strong binding to P. aeruginosa cell surface. The ability of the phage clones to interact specifically with P. aeruginosa was demonstrated by using enzyme-linked immunosorbent assay (ELISA). We assessed selectivity of phage-bacteria-binding by comparing the binding ability of the selected clones to the selector bacterium and a panel of other bacterial species; we also demonstrated by dot spot and immunoblotting that the most reactive and selective phage peptide bound with high avidity the bacterial cell surface. In addition, as proof-of-concept, we tested the possibility to immobilize the affinity-selected phage to a putative biosensor surface. The quality of phage deposition was monitored by ELISA, and phage-bacterial-binding was confirmed by high-power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including clinical-based diagnostics and possibly biological warfare applications.

Identification of a human immunodominant B-cell epitope within the immunoglobulin A1 protease of Streptococcus pneumoniae.

BACKGROUND: The IgA1 protease of Streptococcus pneumoniae is a proteolytic enzyme that specifically cleaves the hinge regions of human IgA1, which dominates most mucosal surfaces and is the major IgA isotype in serum. This protease is expressed in all of the known pneumococcal strains and plays a major role in pathogen's resistance to the host immune response. The present work was focused at identifying the immunodominant regions of pneumococcal IgA1 protease recognized by the human antibody response. RESULTS: An antigenic sequence corresponding to amino acids 420-457 (epiA) of the iga gene product was identified by screening a pneumococcal phage display library with patients' sera. The epiA peptide is conserved in all pneumococci and in two out of three S. mitis strains, while it is not present in other oral streptococci so far sequenced. This epitope was specifically recognized by antibodies present in sera from 90% of healthy adults, thus representing an important target of the humoral response to S. pneumoniae and S. mitis infection. Moreover, sera from 68% of children less than 4 years old reacted with the epiA peptide, indicating that the human immune response against streptococcal antigens occurs during childhood. CONCLUSION: The broad and specific recognition of the epiA polypeptide by human sera demonstrate that the pneumococcal IgA1 protease contains an immunodominant B-cell epitope. The use of phage display libraries to identify microbe or disease-specific antigens recognized by human sera is a valuable approach to epitope discovery.

Efficient display of scFv antibodies on bacteriophage lambda.

In the present work we demonstrate the efficient display of functional scFv antibodies on the bacteriophage lambda capsid. A single-chain (scFv) anti-CEA antibody gene was cloned in two different vectors to obtain fusion of the scFv antibody to the N- or C-terminus of the bacteriophage lambda capsid protein D (gpD). Lambda bacteriophage assembly occurs in the reducing environment of the cytoplasm; despite this the lambda-displayed anti-CEA antibody fragments retain the capacity to recognize the antigen, indicating correct single-chain antibody folding. Efficient production of functional scFv exposed on lambda capsid with viable antigen binding specificity allowed us to study and compare the capacity of display, the stability of recombinant antibody expression and the assembly efficiency of bacteriophage particles decorated with recombinant antibody fused to the amino- or carboxy-terminus of lambda D protein.

Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein.

BACKGROUND: CEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration. METHODS: The human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, by in vitro mutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized. RESULTS: The scFv MA39 antibody was affinity-maturated by in vitro mutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized for expression in a large panel of human normal and tumor tissues and cells. CONCLUSION: The binding affinity of the scFv E8 is in a range for efficient, in vivo, antigen capture in tumor cells expressing a shared epitope of the CEACAM1, 3 and 5 proteins. This new immunoreagent meets all criteria for a potential anticancer compound: it is human, hence poorly or not at all immunogenic, and it binds selectively and with good affinity to the CEA epitope expressed by metastatic melanoma and colon and lung carcinomas. Furthermore, its small molecular size should provide for efficient tissue penetration, yet give rapid plasma clearance.

Discovery of novel Streptococcus pneumoniae antigens by screening a whole-genome lambda-display library.

Streptococcus pneumoniae is a causative agent of otitis media, pneumonia, meningitis and sepsis in humans. For the development of effective vaccines able to prevent pneumococcal infection, characterization of bacterial antigens involved in host immune response is crucial. In order to identify pneumococcal proteins recognized by host antibody response, we created an S. pneumoniae D39 genome library, displayed on lambda bacteriophage. The screening of such a library, with sera either from infected individuals or mice immunized with the S. pneumoniae D39 strain, allowed identification of phage clones carrying S. pneumoniae B-cell epitopes. Epitope-containing fragments within the families of the histidine-triad proteins (PhtE, PhtD), the choline-binding proteins (PspA, CbpD) and zinc metalloproteinase B (ZmpB) were identified. Moreover, library screening also allowed the isolation of phage clones carrying three distinct antigenic regions of a hypothetical pneumococcal protein, encoded by the ORF spr0075 in the R6 strain genome sequence. In this work, Spr0075 is first identified as an expressed S. pneumoniae gene product, having an antigenic function during infection.

Identification and refinement of a peptide affinity ligand with unique specificity for a monoclonal anti-tenascin-C antibody by screening of a phage display library.

Using phage display technology, a 22-mer peptide was selected as a ligand with unique specificity for the murine monoclonal ST2146 antibody that recognizes the EGF repeats region of the human tumor-associated antigen tenascin-C. This peptide, synthesized in an 8-branched form to enhance its binding properties, is useful in replacing the native antigen in the affinity and immunoreactivity characterization of the ST2146 antibody and its biotinylated derivatives. Affinity resins, prepared by immobilizing the mimotope or its shorter 10-mer binding unit on a chromatographic support, were able to capture ST2146 directly from the hybridoma supernatant, with antibody recovery and host cell protein (HCP) reduction similar to or better than protein A sorbent, a purity degree exceeding 95%, and full recovery of antibody activity. The affinity constants of both peptides, as determined by frontal analysis of broad-zone elution affinity chromatography and BiaCore measurements, were very similar and included in a range suitable for affinity ligands. Column capacity, determined by applying a large excess of purified ST2146 to 1 mL of column bed volume, was close to 50 mg/mL for both resins. These matrices retain their ST2146 binding properties after various treatments, including sanitization, thus indicating very high stability in terms of ligand leakage and degradation. Moreover, the short form shows higher enzymatic stability, thus proving more suitable as ligand for ST2146 affinity purification.

Functional characterization of a monoclonal antibody epitope using a lambda phage display-deep sequencing platform.

We have recently described a method, named PROFILER, for the identification of antigenic regions preferentially targeted by polyclonal antibody responses after vaccination. To test the ability of the technique to provide insights into the functional properties of monoclonal antibody (mAb) epitopes, we used here a well-characterized epitope of meningococcal factor H binding protein (fHbp), which is recognized by mAb 12C1. An fHbp library, engineered on a lambda phage vector enabling surface expression of polypeptides of widely different length, was subjected to massive parallel sequencing of the phage inserts after affinity selection with the 12C1 mAb. We detected dozens of unique antibody-selected sequences, the most enriched of which (designated as FrC) could largely recapitulate the ability of fHbp to bind mAb 12C1. Computational analysis of the cumulative enrichment of single amino acids in the antibody-selected fragments identified two overrepresented stretches of residues (H248-K254 and S140-G154), whose presence was subsequently found to be required for binding of FrC to mAb 12C1. Collectively, these results suggest that the PROFILER technology can rapidly and reliably identify, in the context of complex conformational epitopes, discrete "hot spots" with a crucial role in antigen-antibody interactions, thereby providing useful clues for the functional characterization of the epitope.

Phage display revisited: Epitope mapping of a monoclonal antibody directed against Neisseria meningitidis adhesin A using the PROFILER technology.

There is a strong need for rapid and reliable epitope mapping methods that can keep pace with the isolation of increasingly larger numbers of mAbs. We describe here the identification of a conformational epitope using Phage-based Representation OF ImmunoLigand Epitope Repertoire (PROFILER), a recently developed high-throughput method based on deep sequencing of antigen-specific lambda phage-displayed libraries. A novel bactericidal monoclonal antibody (mAb 9F11) raised against Neisseria meningitidis adhesin A (NadA), an important component of the Bexsero(®) anti-meningococcal vaccine, was used to evaluate the technique in comparison with other epitope mapping methods. The PROFILER technology readily identified NadA fragments that were capable of fully recapitulating the reactivity of the entire antigen against mAb 9F11. Further analysis of these fragments using mutagenesis and hydrogen-deuterium exchange mass-spectrometry allowed us to identify the binding site of mAb 9F11 (A250-D274) and an adjoining sequence (V275-H312) that was also required for the full functional reconstitution of the epitope. These data suggest that, by virtue of its ability to detect a great variety of immunoreactive antigen fragments in phage-displayed libraries, the PROFILER technology can rapidly and reliably identify epitope-containing regions and provide, in addition, useful clues for the functional characterization of conformational mAb epitopes.

Epitope Mapping of a Monoclonal Antibody Directed against Neisserial Heparin Binding Antigen Using Next Generation Sequencing of Antigen-Specific Libraries.

We explore here the potential of a newly described technology, which is named PROFILER and is based on next generation sequencing of gene-specific lambda phage-displayed libraries, to rapidly and accurately map monoclonal antibody (mAb) epitopes. For this purpose, we used a novel mAb (designated 31E10/E7) directed against Neisserial Heparin-Binding Antigen (NHBA), a component of the anti-group B meningococcus Bexsero® vaccine. An NHBA phage-displayed library was affinity-selected with mAb 31E10/E7, followed by massive sequencing of the inserts present in antibody-selected phage pools. Insert analysis identified an amino acid stretch (D91-A128) in the N-terminal domain, which was shared by all of the mAb-enriched fragments. Moreover, a recombinant fragment encompassing this sequence could recapitulate the immunoreactivity of the entire NHBA molecule against mAb 31E10/E7. These results were confirmed using a panel of overlapping recombinant fragments derived from the NHBA vaccine variant and a set of chemically synthetized peptides covering the 10 most frequent antigenic variants. Furthermore, hydrogen-deuterium exchange mass-spectrometry analysis of the NHBA-mAb 31E10/E7 complex was also compatible with mapping of the epitope to the D91-A128 region. Collectively, these results indicate that the PROFILER technology can reliably identify epitope-containing antigenic fragments and requires considerably less work, time and reagents than other epitope mapping methods.

Peptides mimicking Vibrio cholerae O139 capsular polysaccharide elicit protective antibody response.

Vibrio cholerae is the etiological agent of cholera. V. cholerae serogroup O1 had been, until 1992, the only serogroup responsible for large epidemics and pandemics of cholera. In 1992, a new serotype of V. cholerae emerged in South-East Asia that caused a massive outbreak of cholera in India and neighboring countries. The new serotype was named V. cholerae O139. The main differences between V. cholerae O139 and O1 are that the former possesses a capsular polysaccharide and different lipopolysaccharide. Capsular polysaccharides are, in general, T-independent antigens giving rise to poor immune responses lacking immunological memory. In order to overcome this, monoclonal antibodies against the capsular polysaccharide of V. cholerae O139 were used to screen different phage-displayed random peptide libraries. Eight different phage clones were selected and characterized using enzyme immunoassay with the monoclonal antibodies, and then tested for specificity by competition with V. cholerae O139 capsular polysaccharide. Selected peptides were sequenced, synthesized and conjugated to bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). The conjugated peptides were used to immunize mice. It is evident that the anti-peptide mouse antibodies bind to the V. cholerae O139 capsular polysaccharide. In addition, the anti-peptide antibodies are protective in a suckling mouse model. The protective efficacy is both specific and dose-dependent. A PCT (PCT/IT2003/000489) with the publication number WO 2004/056851 has been filed for the sequences of the eight peptides.

Display libraries on bacteriophage lambda capsid.

Phage display is an established technology that has been successfully applied, in the last fifteen years, to projects aimed at deciphering biological processes and/or at the isolation of molecules of practical value in several diverse applications. Bacteriophage lambda, representing a molecular cloning and expression tool widely utilized since decades, has also been exploited to develop vectors for the display of libraries on its capsid. In the last few years, lambda display approach has been consistently offering new enthralling perspectives of technological application, such as domain mapping, antigen discovery, and protein interaction studies or, more generally, in functional genomics.

Determination of the fine epitope specificity of an anti-hepatitis B virus X protein monoclonal antibody using microanalytical and molecular biological methods.

The recombinant form of the 17kDa, highly hydrophobic and disulfide-bonded hepatitis B virus X protein (HBX) was used for developing a set of monoclonal antibodies (Mab). Our present goal was to determine the fine epitope specificity of our anti-HBX Mab. Based on computer analysis two sequences (amino acids 22-31 and 100-114) were predicted for possessing high immunogenity while the anti-HBX Mab did not recognized them. Limited proteolysis and mass spectroscopic analysis suggested another possible sequence (amino acids 14-26), which also proved to be negative using an immunoserological test. Subsequently, we performed a screen of a phage displayed random peptide library, by which we could localize the epitope to amino acids 88-93. This finding was confirmed using three overlapping fusion peptides spanning amino acids 77-142. Their testing in ELISA assigned the epitope to amino acids 77-95, which supports the result obtained by screening the phage displayed library. Our results suggest the necessity of a complex application of current molecular biological and immunological techniques in fine structure mapping. This approach will be useful to study the prognostic relevance of different antigenic sites on HBX during the development of chronic hepatitis and primary hepatocellular carcinoma.

Selection of mimotopes of the cell surface adhesion molecule Mel-CAM from a random pVIII-28aa phage peptide library.

The cell surface adhesion molecule Mel-CAM is highly expressed in advanced primary and metastatic melanoma. Mel-CAM was first described as an integral membrane glycoprotein of malignant melanoma cells. The murine monoclonal antibody MAd18-5D7 recognizes an epitope of the extracellular domain of Mel-CAM and is able to enhance Mel-CAM mediated adhesion of melanoma cells in aggregation assays. For the characterization of peptides that antigenically mimic surface-exposed areas of Mel-CAM we screened a newly constructed random pVIII-28aa bacteriophage peptide library against MAd18-5D7. After three panning rounds a population of phages binding to MAd18-5D7 was enriched. Peptides expressed on the surface of these phages were then tested for their specificity for the antibody's antigen binding site. DNA sequences coding for two specific peptide ligands were determined. One of the deduced amino acid sequences showed similarity to a portion of the sequence of the third immunoglobulin-like extracellular domain of Mel-CAM. Both peptides blocked the interaction of MAd18-5D7 with Mel-CAM present in a MelJuSo melanoma cell line lysate. Phage displayed as well as synthetic peptides inhibited in a dose-dependent manner the binding of MAd18-5D7 to recombinant Mel-CAM in enzyme-linked immunosorbent assay experiments. No such inhibition was observed using a panel of other anti-Mel-CAM antibodies. Our results clearly indicate that these 28mer peptides are structural equivalents of the MAd18-5D7 epitope of Mel-CAM and that they will be useful tools for further in vitro and in vivo studies of Mel-CAM mediated cell-cell interaction.

The Toxoplasma gondii bradyzoite antigens BAG1 and MAG1 induce early humoral and cell-mediated immune responses upon human infection.

Infection of humans by Toxoplasma gondii leads to an acute systemic phase, in which tachyzoites disseminate throughout the body, followed by a chronic phase characterized by the presence of tissue cysts, containing bradyzoites, in brain, heart and skeletal muscles. This work focused on studying the antigenic regions of bradyzoite-specific proteins involved in human B- and T-cell responses. To this aim, we constructed a phage-display library of DNA fragments derived from the bradyzoite-specific genes BAG1, MAG1, SAG2D, SAG4, BSR4, LDH2, ENO1 and p-ATPase. Challenge of the bradyzoite library with sera of infected individuals led to the identification of antigenic regions within BAG1 and MAG1 gene products. Analysis of the humoral and lymphoproliferative responses to recombinant antigens demonstrated that the BAG1 fragment induced T-cell proliferation in 34% of T. gondii-exposed individuals, while 50% of them had specific IgG. In the same subjects, the MAG1 fragment was recognized by T cells from 17% of the exposed donors and by antibodies from 73% of them. A detailed analysis of the antibody response against BAG1 and MAG1 antigen fragments demonstrated that the immune response against bradyzoites occurs early after infection in humans. Finally, we provide evidence that the T-cell response against BAG1 is associated with the production of interferon-gamma, suggesting that bradyzoite antigens should be considered in the design of potential vaccines in humans.

Molecular dissection of the human B-cell response against Toxoplasma gondii infection by lambda display of cDNA libraries.

The disorders generated by Toxoplasma gondii infection are closely associated with the competence of the host immune system and both humoral and cell mediated immunity are involved in response to parasite invasion. To identify antigens implicated in human B-cell responses, we screened a phage-display library of T. gondii cDNA fragments with sera of infected individuals. This approach identified a panel of recombinant phage clones carrying B-cell epitopes. All the peptide sequences selected by this procedure are regions of T. gondii gene products. These regions contain epitopes of the T. gondii antigens SAG1, GRA1, GRA7, GRA8 and MIC5, which are recognised by human immunoglobulins. Moreover, we report the isolation and characterisation of two additional immunodominant regions encoded by GRA3 and MIC3 genes, whose products have never been described as antigens of the human B-cell response against T. gondii infection. These results demonstrate potential of lambda-display technology for antigen discovery and for the study of the human antibody response against infectious agents.