Tetanus neurotoxin-insensitive vesicle-associated membrane protein localizes to a presynaptic membrane compartment in selected terminal subsets of the rat brain.

Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is a vesicular soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor (SNARE) that has been implicated in neurite outgrowth. It has previously been reported that TI-VAMP is localised in the somatodendritic compartment of neurons indicating a role in membrane fusion events within dendrites. Using a newly produced monoclonal antibody to TI-VAMP that improves signal/noise immunodetection, we report that TI-VAMP is also present in subsets of axon terminals of the adult rat brain. Four distinctive populations of labelled axon terminals were identified: 1) the hippocampal mossy fibres of the dentate gyrus and of CA3, 2) the striatal peridendritic terminal plexuses in the globus pallidus (GP), substantia nigra pars reticulata (SNr), 3) peridendritic plexuses in the central nucleus of the amygdala, and 4) the primary sensory afferents in the dorsal horn of the spinal cord. The presynaptic localisation of TI-VAMP in these locations was demonstrated by co-localisation with synaptophysin. Ultrastructural studies showed TI-VAMP labelling over synaptic vesicles in the mossy fibres, whereas it was localised in tubulo-vesicular structures and multivesicular bodies in the pyramidal cell dendrites. The presynaptic localisation of TI-VAMP occurred by P15, so relatively late during development. In contrast, dendritic labelling was most prominent during the early post-natal period. Co-localisation with markers of neurotransmitters showed that TI-VAMP-positive terminals are GABAergic in the GP and SNr and glutamatergic in the mossy fibre system and in the dorsal root afferents. Most of these terminals are known to co-localise with neuropeptides. We found met-enkephalin-immunoreactivity in a sizeable fraction of the TI-VAMP positive terminals in the GP, amygdala, and dorsal horn, as well as in a few mossy fibre terminals. The function of TI-VAMP in subsets of mature axon terminals remains to be elucidated; it could participate in the exocytotic molecular machinery and/or be implicated in particular growth properties of the mature axon terminals. Thus, the presence of TI-VAMP in the mossy fibres may correspond to the high degree of plasticity that characterises this pathway throughout adult life.

Inhibition of potato virus Y NIa activity: preparation of monoclonal antibody directed against PVY NI protein that inhibits cleavage of PVY polyprotein.

A partially purified nuclear inclusion (NI) fraction was obtained from tobacco plants infected by potato virus Y (PVY). Four monoclonal antibodies (MAbs) were produced and characterized using this semipurified fraction as antigen. Data showed that only one was directed against NIa whereas two were directed against cytoplasmic inclusion (CI) protein and the last one against coat protein (CP). These results were due to the fact that the semipurified NI fraction was usually contaminated with CI and CP proteins. When used on in situ immunofluorescence method the anti-NIa MAb showed accumulation of the NIa protein in both nucleus and cytoplasm. In vivo, this MAb was able to detect different forms of the NIa protein including precursors and cleavage products. It was also able to inhibit the cleavage of the polyprotein detected in the semipurified NI.

Calreticulin, a potential cell surface receptor involved in cell penetration of anti-DNA antibodies.

A 50-kDa protein was purified as a potential receptor, using an affinity matrix containing biotinylated F14.6 or H9.3 anti-DNA mAbs derived from autoimmune (New Zealand Black x New Zealand White)F(1) mouse and membrane extracts from cells. This protein was identified as calreticulin (CRT) by microsequencing. Confocal microscopy and FACS analysis showed that CRT was present on the surface of various cells. CRT protein was recognized by a panel of anti-DNA mAbs in ELISA. The binding of F14.6 to lymphocytes and Chinese hamster ovary cells was inhibited by soluble CRT or SPA-600. Thus, the anti-DNA mAbs used in this study bound to CRT, suggesting that CRT may mediate their penetration into the cells and play an important role in lupus pathogenesis.

Crystal structure of an anti-carbohydrate antibody directed against Vibrio cholerae O1 in complex with antigen: molecular basis for serotype specificity.

The crystal structure of the murine Fab S-20-4 from a protective anti-cholera Ab specific for the lipopolysaccharide Ag of the Ogawa serotype has been determined in its unliganded form and in complex with synthetic fragments of the Ogawa O-specific polysaccharide (O-SP). The upstream terminal O-SP monosaccharide is shown to be the primary antigenic determinant. Additional perosamine residues protrude outwards from the Ab surface and contribute only marginally to the binding affinity and specificity. A complementary water-excluding hydrophobic interface and five Ab-Ag hydrogen bonds are crucial for carbohydrate recognition. The structure reported here explains the serotype specificity of anti-Ogawa Abs and provides a rational basis toward the development of a synthetic carbohydrate-based anti-cholera vaccine.

Combining phage display and molecular modeling to map the epitope of a neutralizing antitoxin antibody.

Crotoxin is a potent presynaptic neurotoxin from the venom of the rattlesnake Crotalus durissus terrificus. It is composed of the noncovalent and synergistic association of a weakly toxic phospholipase A2, CB, and a nontoxic three-chain subunit, CA, which increases the lethal potency of CB. The A-56.36 mAb is able to dissociate the crotoxin complex by binding to the CA subunit, thereby neutralizing its toxicity. Because A-56.36 and CB show sequence homology and both compete for binding to CA, we postulated that A-56.36 and CB had overlapping binding sites on CA. By screening random phage-displayed libraries with the mAb, phagotopes bearing the (D/S)GY(A/G) or AAXI consensus motifs were selected. They all bound A-56.36 in ELISA and competed with CA for mAb binding, although with different reactivities. When mice were immunized with the selected clones, polyclonal sera reacting with CA were induced. Interestingly, the raised antibodies retained the crotoxin-dissociating effect of A-56.36, suggesting that the selected peptides may be used to produce neutralizing antibodies. By combining these data with the molecular modeling of CA, it appeared that the functional epitope of A-56.36 on CA was conformational, one subregion being discontinuous and corresponding to the first family of peptides, the other subregion being continuous and composed of amino acids of the second family. Phage-displayed peptides corresponding to fragments of the two identified regions on CA reacted with A-56.36 and with CB. Our data support the hypothesis that A-56.36 and CB interact with common regions of CA, and highlight residues which are likely to be critical for CA-CB complex formation.

Crystal structure of the allergen Equ c 1. A dimeric lipocalin with restricted IgE-reactive epitopes.

The three-dimensional structure of the major horse allergen Equ c 1 has been determined at 2.3 A resolution by x-ray crystallography. Equ c 1 displays the typical fold of lipocalins, a beta-barrel flanked by a C-terminal alpha-helix. The space between the two beta-sheets of the barrel defines an internal cavity that could serve, as in other lipocalins, for the binding and transport of small hydrophobic ligands. Equ c 1 crystallizes in a novel dimeric form, which is distinct from that observed in other lipocalin dimers and corresponds to the functional form of the allergen. Binding studies of point mutants of the allergen with specific monoclonal antibodies raised in mouse and IgE serum from horse allergic patients allowed to identify putative B cell antigenic determinants. In addition, total inhibition of IgE serum recognition by a single specific monoclonal antibody revealed the restricted nature of the IgE binding target on the molecular surface of Equ c 1.

Identification of a peptide blocking vascular endothelial growth factor (VEGF)-mediated angiogenesis.

Vascular endothelial growth factor (VEGF) binding to the kinase domain receptor (KDR/FLK1 or VEGFR-2) mediates vascularization and tumor-induced angiogenesis. Since there is evidence that KDR plays an important role in tumor angiogenesis, we sought to identify peptides able to block the VEGF-KDR interaction. A phage epitope library was screened by affinity for membrane-expressed KDR or for an anti-VEGF neutralizing monoclonal antibody. Both strategies led to the isolation of peptides binding KDR specifically, but those isolated by KDR binding tended to display lower reactivities. Of the synthetic peptides corresponding to selected clones tested to determine their inhibitory activity, ATWLPPR completely abolished VEGF binding to cell-displayed KDR. In vitro, this effect led to the inhibition of the VEGF-mediated proliferation of human vascular endothelial cells, in a dose-dependent and endothelial cell type-specific manner. Moreover, in vivo, ATWLPPR totally abolished VEGF-induced angiogenesis in a rabbit corneal model. Taken together, these data demonstrate that ATWLPPR is an effective antagonist of VEGF binding, and suggest that this peptide may be a potent inhibitor of tumor angiogenesis and metastasis.

Neutralizing human anti crotoxin scFv isolated from a nonimmunized phage library.

Combinatorial phage display technology offers a new possibility for making human antibodies which could be used in immune therapy. We explored the use of this technology to make human scFvs specific for crotoxin, the main toxic component of the venom of the South-American rattlesnake Crotalus durissus terrificus. Crotoxin, a phospholipase A2 neurotoxin constituted by the association of two subunits, exerts its lethal action by blocking neuromuscular transmission. This is the first report of human anticrotoxin scFvs (scFv 1, scFv 6 and scFv 8) isolated from a naive library of more than 1010 scFv clones with in vivo neutralizing activity. Nevertheless, differences are observed at the level of biological and immunological effects. Only scFv 8 is able to reduce the myotoxicity induced by crotoxin and scFv 1 is capable of altering the in vitro enzymatic activity of this toxin. All three scFvs recognize a region of one subunit located at the junction with the other one. Moreover these scFvs share strong amino acid homologies at the level of either the heavy or the light chain. Taken together, our results suggest that the use of human anticrotoxin scFvs may lead to a new and less aggressive passive immune therapy against poisoning by the venom of Crotalus durissus terrificus.

The 213-amino-acid leucine-rich repeat region of the listeria monocytogenes InlB protein is sufficient for entry into mammalian cells, stimulation of PI 3-kinase and membrane ruffling.

The Listeria monocytogenes InlB protein is a 630-amino-acid surface protein that mediates entry of the bacterium into a wide variety of cell types, including hepatocytes, fibroblasts and epithelial cells such as Vero, HEp-2 and HeLa cells. Invasion stimulates host proteins tyrosine phosphorylation, PI 3-kinase activity and rearrangements in the actin cytoskeleton. We previously showed that InlB is sufficient for entry of InlB-coated latex beads into cells and recent results indicate that purified InlB can stimulate PI 3-kinase activity and is thus the first bacterial agonist of this lipid kinase. In this study, we identified the region of InlB responsible for entry and stimulation of signal transduction events. Eight monoclonal antibodies directed against InlB were raised and, of those, five inhibited bacterial entry. These five antibodies recognized epitopes within the leucine-rich repeat (LRR) region and/or the inter-repeat (IR) region. InlB-staphylococcal protein A (SPA) fusion proteins and recombinant InlB derivatives were generated and tested for their capacity to mediate entry into cultured mammalian cells. All the InlB derivatives that carried the amino-terminal 213-amino-acid LRR region conferred invasiveness to the normally non-invasive bacterium L. innocua or to inert latex beads and the corresponding purified polypeptides inhibited bacterial entry. In addition, the 213-amino-acid LRR region was able to stimulate PI 3-kinase activity and changes in the actin cytoskeleton (membrane ruffling). These properties were not detected with purified internalin, another invasion protein of L. monocytogenes that displays LRRs similar to those of InlB. Taken together, these results show that the first 213 amino acids of InlB are critical for its specific properties.

The 7alpha-hydroxysteroids produced in human tonsils enhance the immune response to tetanus toxoid and Bordetella pertussis antigens.

Human tonsils were assessed for their ability to 7alpha-hydroxylate pregnenolone (PREG), dehydroepiandrosterone (DHEA) and 3-epiandrosterone (EPIA). Both 7alpha-hydroxy-DHEA and 7alpha-hydroxy-EPIA were produced by homogenates of either whole tonsils or of lymphocyte-depleted tonsil fractions. In contrast, isolated lymphocytes were found to be unable to carry out 7alpha-hydroxylation. When co-cultures of tonsil-derived T and B lymphocytes were set up under stimulatory conditions, IgGs were released in the supernatants and could be quantitated, and immunomodulating properties of different steroids were monitored. When PREG was added to a mixture of tonsil-derived B and T lymphocytes, a decrease of non-specific and specific IgG was observed. An increase in specific anti-tetanus toxoid and anti-Bordetella pertussis antigen IgGs was obtained with either 1 microM 7alpha-hydroxy-DHEA or 1 microM 7alpha-hydroxy-EPIA. In contrast, DHEA and EPIA were unable to trigger such an effect. When cultures of isolated tonsillar B cells were used, none of the steroids tested showed significant effects on specific IgG productions. These data led to the conclusion that human tonsillar cells transform DHEA and EPIA, but not PREG, into 7alpha-hydroxylated metabolites. These metabolites could act on target tonsillar T lymphocytes which in turn act upon B lymphocytes for increasing specific IgG production.

Molecular mimicry between a monoclonal antibody and one subunit of crotoxin, a heterodimeric phospholipase A2 neurotoxin.

Crotoxin is a heterodimeric phospholipase A2 neurotoxin formed by the non-covalent association of an acidic and non-toxic subunit, CA, and a basic and weakly toxic phospholipase A2, CB. The two subunits behave in a synergistic manner. CA enhances the lethal potency of CB by increasing its selectivity of action. The mAb A-56.36, directed against the non-toxic subunit CA, was previously shown to neutralize crotoxin toxicity by dissociating the crotoxin complex. In the present report, a polypeptide sequence similarity was observed between some CDRs of mAb A-56.36 and two regions of CB (pos. 60-80 and 95-110). Phage displayed peptides corresponding to VH2 and VH3 of mAb A-56.36 and to their homologous sequences in CB bind CA to different extents. This observation shows that mAb A-56.36 interacts with a region of CA involved in its interaction with CB, therefore mimicking the binding of CB to CA. A similar approach was used to determine the regions of ammodytoxin A and of agkistrodotoxin, two phospholipase A2 neurotoxins similar to CB, which are involved in the formation of heterocomplexes with CA. The analysis of these data contributes to the determination of stretches of amino acids which could constitute the paratope of mAb A-56.36, as well as the region of association of CB with CA in crotoxin.

A recombinant Fab neutralizes dengue virus in vitro.

A recombinant Fab that recognizes a neutralizing epitope located in the (296-400) region of protein E of dengue virus was obtained from cloned hybridoma cells secreting the mouse monoclonal antibody (mAb) 4E11. The Fd and light chain antibody genes were amplified by polymerase chain reaction, cloned into the phagemid vector pMad, expressed in bacteria to produce Fab fragments and sequenced. The mAb 4E11, in particular its light chain complementary-determining regions, shared homologies with two other anti-viral mAbs. The affinity of the parental mAb and the cloned Fab to the MalE-E(296-400) fusion protein were shown to be of the same magnitude, i.e. nanomolar. Fab 4E11 neutralization capacity was found between 8 and 4-times or less lower than that of mAb 4E11, depending on serotypes, thus the Fab could have a smaller antiviral activity than the mAb in vitro.

Immunochemical analysis of UMP kinase from Escherichia coli.

Mono- and polyclonal antibodies directed against UMP kinase from Escherichia coli were tested with the intact protein or with fragments obtained by deletion mutagenesis. As detected in enzyme-linked immunosorbent assay tests, the carboxy-terminal quarter of UMP kinase is immunodominant. Polyclonal antibodies inhibited the enzyme activity with partial or total loss of allosteric effects exerted by UTP and GTP, respectively. These data indicate that the UTP and GTP binding sites in UMP kinase are only partially overlapping. One monoclonal antibody (44-2) recognized a linear epitope in UMP kinase between residues 171 and 180. A single substitution (D174N) in this segment of the enzyme abolished its interaction with the monoclonal antibody (44-2). Polyclonal antisera were used to identify UMP kinase in the bacterial proteome. The enzyme appears as a single spot on two-dimensional electrophoresis at a pI of 7.24 and an apparent molecular mass of 26 kDa. Immunogold labeling of UMP kinase in whole E. coli cells shows a localization of the protein near the bacterial membranes. Because the protein does not contain sequences usually required for compartmentalization, the aggregation properties of UMP kinase observed in vitro might play a role in this phenomenon. The specific localization of UMP kinase might also be related to its putative role in cell division.

A monoclonal antibody directed against the non-toxic subunit of a dimeric phospholipase A2 neurotoxin, crotoxin, neutralizes its toxicity.

Crotoxin is the main toxic component of the venom of the South-American rattlesnake Crotalus durissus terrificus. It is a phospholipase A2 neurotoxin constituted by the association of two subunits: an acidic, non-toxic and non-enzymatic subunit (CA) and a basic, weakly toxic phospholipase A2 (CB). A murine monoclonal antibody directed to the non-toxic subunit CA, A-56.36, was shown to fully neutralize the toxicity of crotoxin. When the in vitro pharmacological properties of crotoxin were further tested, A-56.36 was shown to enhance the enzymatic activity on negatively-charged phospholipids and to increase the acetylcholine release triggered by crotoxin on Torpedo synaptosomes. These effects were explained by the fast dissociation of the crotoxin complex in the presence of the monoclonal antibody A-56.36 and the immunocomplexation of CA, with CB being released in solution. CB is less toxic than crotoxin, has a higher enzymatic activity and triggers a higher acetylcholine release than crotoxin, due to its strong enzymatic activity. A single-chain variable fragment antibody was prepared from monoclonal antibody A-56.36. It binds to CA with a similar affinity than the parental immunoglobulin and exhibits similar effects on the in vitro pharmacological properties of crotoxin.

Phage-displayed mimotopes elicit monoclonal antibodies specific for a malaria vaccine candidate.

The phage-displayed peptide CGRVCLRC (C15) has been isolated from a random library by affinity screening with the D14-3 monoclonal antibody, which was raised to the 42 kDa C-terminal fragment of the major merozoite surface protein 1 of Plasmodium vivax (Pv42). In order to investigate the use of such mimotopes as possible vaccine components, we studied the antibody response in Biozzi mice immunized with C15. High titers of antibodies cross-reacting with Pv42 were generated and the IC50 of all immune sera were in the 5 x 10(-9) M range. Two monoclonal antibodies that specifically bind the Pv42 fragment were isolated. Although these mAbs had a lower affinity for Pv42 when compared to D14-3, they reproduced the cross-reactivity of D14-3 with the equivalent protein in P. cynomolgi, a close relative of P. vivax. DNA sequence analysis showed similarities between the germline genes and the canonical CDR conformations of all three antibodies, but molecular modeling failed to reveal common structural features of their paratopes that could account for their cross-reacting patterns. These data demonstrate that mimotopes selected from random repertoires do not necessarily represent structural equivalents of the original antigen but provide functional images that could replace it for vaccine development.

Structure/antigenicity relationship of cyclic and linear peptides mimicking the V3 loop of HIV2 envelope glycoprotein.

We report the structure and antigenicity of the third variable region (V3) of the HIV2 envelope glycoprotein by the use of linear and cyclic peptides. To this end, a peptide mimicking this region was synthesized and purified, both as an iodoacetamidated linear peptide and a disulphide-bridged cyclic peptide. The cross-reactivity of three monoclonal antibodies (mAbs) produced against the envelope glycoprotein gp140 with the linear and cyclic peptides was tested with ELISA. The results showed that the cyclic peptide is a better ligand for the 3 mAbs 125-F, 125-J and 125-K. The avidity of the mAb/peptide interaction was further analysed by determining the concentration of linear or cyclic peptide leading to 50% inhibition of mAb-peptide complex formation (K0.5). The K0.5 value of mAb 125-F, which displayed the best reactivity with gp140, was estimated to be 5 times higher for the linear (K0.5 = 1.5 x 10(-6) M) than for the cyclic peptide (K0.5 = 3 x 10(-7) M). This indicates a higher affinity of mAb 125-F for the cyclic peptide. mAb 125-J, which exhibited a lower avidity for the gp140 compared to mAb 125-F, had a similar affinity for the cyclic and the linear peptides (K0.5 = 3 x 10(-7) M). mAb 125-K had the lowest reactivity with gp140 and its binding to adsorbed peptide could not be inhibited by the soluble linear or cyclic peptide used up to 10(-5) M. These results suggest that cyclic peptides may have a higher propensity for adopting a native-like structure for the peptide/antibody interaction. Nuclear magnetic resonance experiments at 25 degrees C in phosphate buffer pH 5.4, however, showed that neither peptide displayed a well-defined structure.

Analysis of human IL-2/IL-2 receptor beta chain interactions: monoclonal antibody H2-8 and new IL-2 mutants define the critical role of alpha helix-A of IL-2.

Interleukin 2 (IL-2) interacts with a receptor (IL-2R) composed of three subunits (IL-2R alpha, IL-2R beta and IL-2R gamma). IL-2R beta plays a critical role in signal transduction. An anti-human IL-2 mAb (H2-8) produced after immunization with peptide 1-30 of IL-2 was found to recognize the region occupied by Asp20, at the exposed interface between alpha-helices A and C. Muteins at position 17 and 20 are not recognized by mAb H2-8. mAb H2-8 specifically inhibits the IL-2 proliferation of TS1beta cells which are dependent on the expression of human IL-2R beta chain for IL-2 proliferation. Substitution at internal position Leu17 demonstrates that this position is essential for IL-2 binding and IL-2 bioactivity. New IL-2 mutants at position Asp20 have been analysed. Substitutions Asp --> Asn, Asp --> Lys, Asp --> Leu, show a correlation between diminished affinity for IL-2 receptor and reduced bioactivity measured on TS1beta cells. Mutein Asp Arg lose affinity for IL-2R and bioactivity simultaneously. Furthermore, during the course of the study we have found that mutein Asp20 --> Leu is an IL-2 antagonist. The biological effects of mAb H2-8 and the properties of new mutants at positions 17 and 20 demonstrate that this region of alpha helix-A is involved in IL-2-IL-2R beta interactions.

Biologically active human anti-crotoxin scFv isolated from a semi-synthetic phage library.

BACKGROUND: The display of repertoires of antibody fragments on the surface of filamentous bacteriophages offers a new way of making antibodies with predefined binding specificities. OBJECTIVES: Here we explored the use of this technology to find human antibodies with biological properties. Phage-scFv specific for crotoxin, the main toxic component of the venom of the South-American rattlesnake Crotalus durissus terrificus, were isolated from a 'single pot' repertoire of more than 10(8) clones made in vitro from human V gene segments [1]. The crotoxin molecule is composed of two noncovalently linked subunits: a basic and weakly toxic phospholipase A2 (PLA2) called component B (CB) and an acidic, nonenzymatic and nontoxic subunit called component A (CA). CA is able to increase the toxicity as well as the specificity of action of CB simultaneously reducing its enzymatic activity. STUDY DESIGN: Two clones were isolated (4-21 and 5-3-1) which are specific of the basic subunit CB, but of a moderate affinity (about 10(-7) M). Clones 4-21 and 5-3-1 have different amino acid sequences and different effects on CB properties suggesting that they are raised against different CB epitopes. Purely cholinergic synaptosomes isolated from Torpedo electric organs provide a suitable model to study the presynaptic effects of crotoxin. In this model, CB was shown to induce a larger acetylcholine release than crotoxin. RESULTS: A dose-dependent increase of acetylcholine release was observed when crotoxin was incubated with increasing amounts of phage-scFv 4-21. This clone was also shown to increase the enzymatic activity of crotoxin. These observations suggest that phage-scFv might dissociate the complex CA-CB. It could be therefore a neutralizing antibody since CB is much less toxic than crotoxin. This shows that 'single pot' libraries are capable of providing not only immunochemical reagents of high specificity but also biological reagents of high quality. The use of this library appears to open new possibilities for immune passive therapy.

Antibodies to the leucine-rich repeat region of internalin block entry of Listeria monocytogenes into cells expressing E-cadherin.

Internalin, a surface protein essential for entry of Listeria monocytogenes EGD into epithelial cells, was used as an antigen to raise nine monoclonal antibodies. These monoclonal antibodies recognized seven distinct epitopes which were located in three different regions of the protein. Three of them inhibited internalin-mediated entry and recognized the amino-terminal leucine-rich repeat region of the protein, suggesting that this region is essential for entry.

Reproducing the immune response against the Plasmodium vivax merozoite surface protein 1 with mimotopes selected from a phage-displayed peptide library.

We have used phage display technology to identify peptides binding D14-3, a monoclonal antibody raised against the M(r) 42,000 C-terminal fragment of Plasmodium vivax merozoite surface protein 1 (PvMSP1). By screening a constrained hexapeptide library, seven independent clones binding D14-3 were isolated. The reactivity of D14-3 for these peptides was lower than for the natural antigen and the antibody binding was strictly associated with the viral context and the peptide conformation. Sequence analysis showed that five of them shared homology with the M(r) 42,000 C-terminal fragment (Pv42) and therefore appears to identify the D14-3 epitope. However, the other two peptides, while related to each other, did not correspond to any sequence in the Pv42 molecules. To evaluate their immunological interest, these phagotopes were injected into mice belonging to Balb/c, IC57BI/6 and Biozzi strains. All animals developed a strong immune response against phage particles but only Biozzi mice produced antibodies cross-reacting with Pv42. All phagotopes in Biozzi mice elicited a specific response against Pv42, even those sharing no sequence similarity with the antigen. Moreover, the avidities of these immune sera and the polyclonal response against Pv42 were comparable, suggesting phagotopes could be used as components of a subunit vaccine based on the C-terminal fragment of MSP1.