PEPOP: computational design of immunogenic peptides.

BACKGROUND: Most methods available to predict protein epitopes are sequence based. There is a need for methods using 3D information for prediction of discontinuous epitopes and derived immunogenic peptides. RESULTS: PEPOP uses the 3D coordinates of a protein both to predict clusters of surface accessible segments that might correspond to epitopes and to design peptides to be used to raise antibodies that target the cognate antigen at specific sites. To verify the ability of PEPOP to identify epitopes, 13 crystallographically defined epitopes were compared with PEPOP clusters: specificity ranged from 0.75 to 1.00, sensitivity from 0.33 to 1.00, and the positive predictive value from 0.19 to 0.89. Comparison of these results with those obtained with two other prediction algorithms showed comparable specificity and slightly better sensitivity and PPV. To prove the capacity of PEPOP to predict immunogenic peptides that induce protein cross-reactive antibodies, several peptides were designed from the 3D structure of model antigens (IA-2, TPO, and IL8) and chemically synthesized. The reactivity of the resulting anti-peptides antibodies with the cognate antigens was measured. In 80% of the cases (four out of five peptides), the flanking protein sequence process (sequence-based) of PEPOP successfully proposed peptides that elicited antibodies cross-reacting with the parent proteins. Polyclonal antibodies raised against peptides designed from amino acids which are spatially close in the protein, but separated in the sequence, could also be obtained, although they were much less reactive. The capacity of PEPOP to design immunogenic peptides that induce antibodies suitable for a sandwich capture assay was also demonstrated. CONCLUSION: PEPOP has the potential to guide experimentalists that want to localize an epitope or design immunogenic peptides for raising antibodies which target proteins at specific sites. More successful predictions of immunogenic peptides were obtained when a peptide was continuous as compared with peptides corresponding to discontinuous epitopes. PEPOP is available for use at http://diagtools.sysdiag.cnrs.fr/PEPOP/.

Mapping the ADF/cofilin binding site on monomeric actin by competitive cross-linking and peptide array: evidence for a second binding site on monomeric actin.

The binding sites for actin depolymerising factor (ADF) and cofilin on G-actin have been mapped by competitive chemical cross-linking using deoxyribonuclease I (DNase I), gelsolin segment 1 (G1), thymosin beta4 (Tbeta4), and vitamin D-binding protein (DbP). To reduce ADF/cofilin induced actin oligomerisation we used ADP-ribosylated actin. Both vitamin D-binding protein and thymosin beta4 inhibit binding by ADF or cofilin, while cofilin or ADF and DNase I bind simultaneously. Competition was observed between ADF or cofilin and G1, supporting the hypothesis that cofilin preferentially binds in the cleft between sub-domains 1 and 3, similar to or overlapping the binding site of G1. Because the affinity of G1 is much higher than that of ADF or cofilin, even at a 20-fold excess of the latter, the complexes contained predominantly G1. Nevertheless, cross-linking studies using actin:G1 complexes and ADF or cofilin showed the presence of low concentrations of ternary complexes containing both ADF or cofilin and G1. Thus, even with monomeric actin, it is shown for the first time that binding sites for both G1 and ADF or cofilin can be occupied simultaneously, confirming the existence of two separate binding sites. Employing a peptide array with overlapping sequences of actin overlaid by cofilin, we have identified five sequence stretches of actin able to bind cofilin. These sequences are located within the regions of F-actin predicted to bind cofilin in the model derived from image reconstructions of electron microscopical images of cofilin-decorated filaments. Three of the peptides map to the cleft region between sub-domains 1 and 3 of the upper actin along the two-start long-pitch helix, while the other two are in the DNase I loop corresponding to the site of the lower actin in the helix. In the absence of any crystal structures of ADF or cofilin in complex with actin, these studies provide further information about the binding sites on F-actin for these important actin regulatory proteins.

The prevalence of proteolytic antibodies against factor VIII in hemophilia A.

BACKGROUND: Factor VIII inhibitors are IgG alloantibodies that arise during replacement therapy in 25 to 50 percent of patients with severe hemophilia A. The hydrolysis of factor VIII by anti--factor VIII antibodies has been proposed as a mechanism of inactivation of factor VIII. METHODS: We purified IgG from patients with severe hemophilia A. The proteolytic activity of the antibodies was assessed by incubating the IgG with biotinylated human factor VIII and analyzing patterns of factor VIII cleavage by sodium dodecyl sulfate--polyacrylamide-gel electrophoresis and immunoblotting. The controls were normal human IgG and IgG purified from plasma of patients with hemophilia who did not have inhibitory antibodies. RESULTS: Significant proteolytic activity was detected in IgG from 13 of 24 inhibitor-positive patients. No hydrolytic activity was detected in control antibodies of IgG from patients without inhibitors. The rate of hydrolysis of factor VIII by purified IgG correlated positively with the factor VIII--neutralizing activity of IgG in plasma (r2=0.67, P=0.029). Principal-component analysis of migration profiles of digestion fragments demonstrated the heterogeneity of the catalytic potential of factor VIII inhibitors among patients. CONCLUSIONS: Proteolysis is a mechanism by which IgG antibodies against factor VIII can inactivate factor VIII.

Cardiac troponin C-L29Q, related to hypertrophic cardiomyopathy, hinders the transduction of the protein kinase A dependent phosphorylation signal from cardiac troponin I to C.

We investigated structural and functional aspects of the first mutation in TNNC1, coding for the calcium-binding subunit (cTnC) of cardiac troponin, which was detected in a patient with hypertrophic cardiomyopathy [ Hoffmann B, Schmidt-Traub H, Perrot A, Osterziel KJ & Gessner R (2001) Hum Mut17, 524]. This mutation leads to a leucine-glutamine exchange at position 29 in the nonfunctional calcium-binding site of cTnC. Interestingly, the mutation is located in a putative interaction site for the nonphosphorylated N-terminal arm of cardiac troponin I (cTnI) [ Finley NL, Abbott MB, Abusamhadneh E, Gaponenko V, Dong W, Seabrook G, Howarth JW, Rana M, Solaro RJ, Cheung HC et al. (1999) EJB Lett453, 107-112]. According to peptide array experiments, the nonphosphorylated cTnI arm interacts with cTnC around L29. This interaction is almost abolished by L29Q, as observed upon protein kinase A-dependent phosphorylation of cTnI at serine 22 and serine 23 in wild-type troponin. With CD spectroscopy, minor changes are observed in the backbone of Ca2+-free and Ca2+-saturated cTnC upon the L29Q replacement. A small, but significant, reduction in calcium sensitivity was detected upon measuring the Ca2+-dependent actomyosin subfragment 1 (actoS1)-ATPase activity and the sliding velocity of thin filaments. The maximum actoS1-ATPase activity, but not the maximum sliding velocity, was significantly enhanced. In addition, we performed our investigations at different levels of protein kinase A-dependent phosphorylation of cTnI. The in vitro assays mainly showed that the Ca2+ sensitivity of the actoS1-ATPase activity, and the mean sliding velocity of thin filaments, were no longer affected by protein kinase A-dependent phosphorylation of cTnI owing to the L29Q exchange in cTnC. The findings imply a hindered transduction of the phosphorylation signal from cTnI to cTnC.

Anti-factor VIII antibodies: a 2005 update.

The development of anti-factor VIII (FVIII) antibodies is currently one of the most serious complications in the treatment of haemophilia A patients. Numerous studies in literature report on their epitope specificity, their mechanism of FVIII inactivation, and their relationship with FVIII genetic alterations. During the last two years, however, a particular effort has been made to better understand their generation, with particular emphasis on the interplay of T cells and B cells specific for FVIII and the generation of anti-FVIII antibodies. Moreover, novel strategies to improve the management or treatment of patients with anti-FVIII antibodies have been recently proposed: the use of less immunogenic engineered recombinant FVIII molecules, neutralization of inhibitors by blocking their deleterious activity either by low molecular weight peptide decoys or by anti-idiotypic antibodies, and attempts to suppress the T-cell response involved in the antibody formation. All of these represent promising therapeutic approaches. This review attempts to sum up current knowledge of the nature and properties of anti-FVIII antibodies, their mechanism of action, their neutralization by anti-idiotypic antibodies, and the role of T cells in FVIII inhibitor formation. In the final part, some of the new strategies susceptible to improve the management or the eradication of anti-FVIII antibodies are presented.

Antibodies with hydrolytic activity towards factor VIII in patients with hemophilia A.

Antibodies endowed with hydrolytic properties have been described in humans for over a decade in a variety of pathological conditions such as systemic lupus erythematosus (SLE), autoimmune thyroiditis, asthma, and Bence Jones disease. Although the identified target substrate molecules have always been autoantigens (i.e., DNA, thyroglobulin, vasoactive intestinal peptide), a direct role of hydrolysis of the autoantigen in pathology of the disease has not been clearly documented. We have described in multitransfused patients with hemophilia A the presence of anti-factor VIII (FVIII) IgG antibodies that hydrolyze FVIII. The estimated kinetic parameters derived for FVIII cleavage by anti-FVIII antibodies are in line with the previously described catalytic antibodies. The identified cleavage sites are evenly spread throughout the FVIII molecule and are located after an arginine or a lysine in most cases. We have recently shown that the catalytic antibodies are highly prevalent among hemophilia A patients with FVIII inhibitors. Catalytic antibodies to FVIII are the first example where the hydrolysis of the target molecule by hydrolytic antibodies may be directly relevant to the etiology of the disease. The characterization of FVIII inhibitors as site-specific proteases may provide novel strategies in the design of therapy against FVIII inhibitors in patients with hemophilia A.

Application of the Spot method to the identification of peptides and amino acids from the antibody paratope that contribute to antigen binding.

Overlapping peptide scans prepared by Spot synthesis have been used to map interaction sites in several systems. Here we report our experience with this approach to identify peptides from the variable parts of anti-hapten, anti-peptide and anti-protein antibodies that retain their specific antigen-binding capacity in the Spot format. In general, the identification by the Spot method of antigen-reactive peptides was confirmed by using soluble peptides which demonstrated antigen-binding capacity in ELISA or Biacore and, biological activity for some peptides derived from anti-CD4 antibodies. The Spot method was also used to map precisely key residues from the antibody paratope. The identification of critical residues from an anti-troponin I antibody of diagnostic interest is reported as well as the compiled results from the analysis of five other antibodies of various specificities. A critical assessment of our results is provided by comparing results obtained by our approach in the mapping of antibody residues critical for antigen binding with data from the literature concerning the structural analysis of antigen-antibody complexes.

Anti-heavy-chain monoclonal antibodies directed to the acidic regions of the factor VIII molecule inhibit the binding of factor VIII to phospholipids and von Willebrand factor.

Recent studies have shown that inhibitors develop against acidic regions of the FVIII molecule, which contain important functional sites. However, their mechanisms of inhibition are not well understood. In this study, two anti-human FVIII mouse monoclonal antibodies (MAbs), directed towards the exposed acidic regions of the FVIII molecule, were developed, characterised and their mechanisms of inhibition investigated. The two MAbs, F7B4 and F26F6, had inhibitory titres of 32 and 944 BU/mg respectively, had high affinities for the FVIII molecule (K(D) approximately nM range) and recognised sequences V(357)-F(360) on the acidic a1 region and E(724)-L(731) on the acidic a2 region of the FVIII heavy-chain (HC), respectively. F7B4 inhibited the rate of FXa generation by activated FVIII, whilst both antibodies inhibited FVIII activation by thrombin and blocked thrombin cleavage of FVIII. Furthermore, F7B4 and F26F6 inhibited FVIII binding to (a) phospholipids (IC(50): 77 nM and 40 nM respectively), and (b) VWF (IC(50): 93 nM and 267 nM respectively), despite both having HC specificity. Experiments with F(ab')(2) fragments confirmed the above findings. Taken together these data represent novel findings in that anti-acidic HC antibodies can inhibit FVIII function by a variety of mechanisms, in particular by interfering with the binding of FVIII to phospholipids & VWF.

Discontinuous epitope prediction based on mimotope analysis.

MOTIVATION: Phage display is a widespread technique used to obtain peptide mimotopes selected by binding to a given monoclonal antibody in a similar way as the native epitope. However, the localization of the interaction site mimicked by the mimotopes on the surface of the antigen is not always a straightforward task. MIMOP is a computational tool developed with the aim of helping experimentalists to analyze a set of mimotope sequences and guide them in the identification of the mimicked region. RESULTS: To predict potential epitopic regions, MIMOP integrates two different approaches combining two- and three-dimensional analyses: MimAlign starts from degenerated alignment analyses, and MimCons is based on consensus identification. The relevance and usefulness of the tool are illustrated by four use cases corresponding to real-life situations.

Assay for measurement of intact B-type natriuretic peptide prohormone in blood.

BACKGROUND: B-Type natriuretic peptide (BNP1-32) as well as the N-terminal fragment of the prohormone containing residues 1-76 (NT-proBNP1-76), both cleavage products of the precursor proBNP1-108, are reported to be powerful markers for prognosis and risk stratification of heart failure. However, the intact precursor also circulates in the bloodstream. Assays for the detection of these cleavage products have been developed, but most of these assays may overestimate the concentrations of the cleavage products because they also measure the precursor form. It is therefore important to develop an immunoassay that specifically measures solely proBNP1-108 in plasma. METHODS: After carefully designing the peptide used to immunize mice, we selected a specific monoclonal antibody (mAb Hinge76) that recognizes the cleavage site of proBNP1-108, an epitope present only in the precursor form. mAb Hinge76 recognizes recombinant proBNP1-108 in a dose-dependent manner, without any significant cross-reactivity with either recombinant NT-proBNP1-76 or synthetic BNP1-32. By combining mAb Hinge76 with a polyclonal antibody directed against BNP1-32, we were able to set up a proBNP1-108-specific sandwich immunoassay able to confirm the presence of proBNP1-108 in blood samples. RESULTS: From a cohort of 50 healthy persons and 170 patients with congestive heart failure (CHF), our assay was able to differentiate healthy individuals from CHF patients (P <0.005). Interestingly, plasma proBNP1-108 concentrations were correlated with New York Heart Association classification. Moreover, a close relationship between proBNP1-108 and BNP1-32 concentrations may exist, as a good correlation (r2= 0.89) was obtained when their respective concentrations were compared. CONCLUSION: mAb Hinge76 is the first proBNP1-108-specific mAb produced that allows accurate estimation of proBNP1-108 concentrations in plasma.

Low molecular weight peptides restore the procoagulant activity of factor VIII in the presence of the potent inhibitor antibody ESH8.

Following repeated administration of factor VIII (FVIII), a significant number of hemophilia A patients develop antibodies (Abs), inhibiting the procoagulant activity of infused FVIII. We have designed an approach based on the blocking of the deleterious activity of these Abs by peptide decoys mimicking the anti-FVIII Ab epitopes. Here, the well characterized inhibitory monoclonal Ab ESH8 served as a model. Several phage peptide libraries were screened for specific binding to ESH8. Seven constrained dodecapeptide sequences were obtained. Six sequences carried the consensus motif, hydrophobic-(Y/F)GKTXL. This motif showed a certain similarity with the (2231)QVDFQKTMKV(2240) sequence of the C(2) domain. In the seventh sequence, YCNPSIGDKNCR, the residues GDKN are similar to the sequence (2267)DGHQ(2270). Upon inspection of the C(2) domain crystallographic structure, the two stretches QVDFQKTMKV and DGHQ appeared close together in space and might constitute a discontinuous epitope. Corresponding synthetic peptides were able to inhibit the binding of ESH8 to FVIII in a specific and dose-dependent manner. Moreover, the ability of the selected peptides to neutralize the inhibitory activity of ESH8 was demonstrated in functional tests as well as in vivo in a murine model of hemophilia A. This study demonstrates the potential of this approach to neutralize the activity of potent inhibitory Abs.

Peptide decoys selected by phage display block in vitro and in vivo activity of a human anti-FVIII inhibitor.

Hemophilia A is a life-threatening, hemorrhagic, X-linked recessive disorder resulting in deficient factor VIII (FVIII) activity. After the infusion of therapeutic FVIII, 25% of patients develop anti-FVIII antibodies that inhibit FVIII procoagulant activity, thus precluding further administration of FVIII. Here we report a novel approach aimed at neutralizing the activity of FVIII inhibitors by peptide epitope surrogates. To illustrate our concept, we chose the human anti-FVIII monoclonal antibody, Bo2C11, as a representative of anti-FVIII antibodies and a phage-displayed peptide library approach to obtain surrogate peptides. We selected a series of constrained dodecapeptides with the core sequence W-NR, which specifically interacts with the combining site of Bo2C11. The peptides mimic the epitope recognized by Bo2C11 and are able to inhibit specifically and in a dose-dependent manner the binding of Bo2C11 to FVIII. Peptide 107, in particular, neutralized the activity of Bo2C11 in vitro and restored normal hemostasis in hemophilic mice. Thus, the use of peptide decoys may be a promising new approach for the neutralization of pathologic antibodies.

Monoclonal antibody 667 recognizes the variable region A motif of the ecotropic retrovirus CasBrE envelope glycoprotein and inhibits Env binding to the viral receptor.

Monoclonal antibody (MAb) 667 is a neutralizing mouse monoclonal antibody recognizing the envelope glycoprotein (Env) of the ecotropic neurotropic murine retrovirus CasBrE but not that of other murine retroviruses. Since 667 can be used for preclinical studies of antiviral gene therapy as well as for studying the early events of retroviral infection, we have cloned its cDNAs and molecularly characterized it in detail. Spot technique-based experiments showed that 667 recognizes a linear epitope of 12 amino acids located in the variable region A of the receptor binding domain. Alanine scanning experiments showed that six amino acids within the epitope are critical for MAb binding. One of them, D(57), is not present in any other murine retroviral Env, which suggests a critical role for this residue in the selectivity of 667. MAb 667 heavy- and light-chain cDNAs were functionally characterized by transient transfection into Cos-7 cells. Enzyme-linked immunosorbent assays and Biacore studies showed that the specificities as well as the antigen-binding thermodynamic and kinetic properties of the recombinant 667 MAb (r667) produced by Cos-7 cells and those of the parental hybridoma-produced MAb (h667) were similar. However, h667 was shown to contain contaminating retroviral and/or retrovirus-like particles which interfere with both viral binding and neutralization experiments. These contaminants could successfully be removed by a stringent purification protocol. Importantly, this purified 667 could completely prevent retrovirus binding to target cells and was as efficient as the r667 MAb produced by transfected Cos-7 cells in neutralization assays. In conclusion, this study shows that the primary mechanism of virus neutralization by MAb 667 is the blocking of the retroviral receptor binding domain of CasBrE Env. In addition, the findings of this study constitute a warning against the direct use of hybridoma cell culture supernatants for studying the initial events of retroviral cell infection as well as for carrying out in vivo neutralization experiments and suggest that either recombinant antibodies or highly purified antibodies are preferable for these purposes.