Cross-reactivity of monoclonal antibodies to a chimeric V3 peptide of HIV-1 with peptide analogues studied by biosensor technology and ELISA.

The reactivity of monoclonal antibodies (Mabs) raised against a cyclic peptide representing a chimeric V3 loop of HIV-1 gp120 with different peptide analogues was studied with a biosensor system (BIAcore) and by ELISA. In both assays, the Mabs cross-reacted extensively with the V3 regions of different HIV-1 strains and recognized the cyclic form of the peptide immunogen better than its linear form. The highest degree of cross-reactivity was observed with peptides that shared a Lys312 with the chimeric sequence. Dissociation rate constants of ten Mabs measured with the BIAcore with respect to different peptides increased with increasing numbers of substitutions in the flanking regions of the V3 tip sequence Gly Pro Gly Arg. Immobilization of the cyclic peptide on the sensor chip via a thiol group added near the end of the loop structure preserved the conformation of the peptide. In view of the good correlation between the BIAcore and ELISA results, biosensor data should be useful for selecting peptides to be used in diagnostic solid phase assays.

Ability of linear and cyclic peptides of neutralization antigenic site 1 of poliovirus type 1 to induce virus cross-reactive and neutralizing antibodies.

Eight peptides encompassing neutralization antigenic site 1 of poliovirus type 1 (residues 93-103 of VP1) were synthesized in linear or cyclized form and used to immunize rabbits. The resulting anti-peptide antibodies were tested for their ability to react with linear peptide 95-104, with infectious virus D-particles and heated C-particles and for their capacity to neutralize poliovirus infectivity. A good correlation was observed between the ability of different peptide antisera to immunoprecipitate D-particles and neutralize virus infectivity. The peptides that induced a neutralizing antibody response in the highest number of immunized animals contained flanking residues 104-115 in addition to the 93-103 residues of the epitope. However, a high neutralizing antibody titre was also obtained in two of ten animals immunized with peptide 93-104 cyclized via an amide bond between Asp93 and Lys103. It seems, therefore, that, at least in rabbits, the T-cell epitope recently identified in residues 103-115 of VP1 need not be present in the peptide immunogen in order to obtain poliovirus-specific neutralizing antibodies.

Selective induction of protection against influenza virus infection in mice by a lipid-peptide conjugate delivered in liposomes.

We have previously reported (Muller et al. Vaccine 1990, 8, 308) that two cyclic peptide analogues called D loop and K loop, corresponding to residues 139-147 in site A of the haemagglutinin (HA) of influenza A virus (strain X31), were both able to provide protective immunity to infected OF1 mice when administered in the form of peptide-ovalbumin conjugates. The predicted conformation of the D loop is nearly identical to that of the native loop known from the X-ray structure of HA, while the predicted conformation of the K loop differs significantly from the native one. In this study, the two peptides were conjugated to small unilamellar liposomes, thus creating a chemically defined immunogen, and OF1 mice were immunized with these liposomes containing monophosphoryl lipid A as adjuvant. Compared with protein carrier systems, the liposomal preparations are completely synthetic and avoid the use of Freund's adjuvant. By using liposomes associated with the D loop, we were able to achieve 70% protection of the mice against intranasal challenge with the influenza virus while no protection was obtained with the liposome-associated K loop. The difference in effect between the two liposome and ovalbumin carrier systems may result from the induction of different structures in the peptides when coupled to lipid anchors than when coupled to proteins.

Cross-reactive potential of rabbit antibodies raised against a cyclic peptide representing a chimeric V3 loop of HIV-1 gp120 studied by biosensor technique and ELISA.

Rabbit antibodies were induced against a free cyclic peptide representing the chimeric sequence of a consensus V3 loop of HIV-1 gp120. The reactivity of these antibodies was tested in a biosensor system (BIAcore, Pharmacia AB, Uppsala, Sweden) and in ELISA with the peptide immunogen in its cyclic and linear forms, as well as with peptides corresponding to the V3 region of different HIV-1 variants. The antibodies reacted with all the peptides tested both in ELISA and in biosensor assays and recognized the cyclic form of the chimeric peptide better than the linear form. Although antibodies raised against the V3 region of particular HIV-1 variants cross-react with other HIV-1 strains, it seems that the use of a chimeric peptide as immunogen improved the cross-reactivity spectrum of recognition of the antibodies. The anti-V3 antibodies were also tested for their ability to neutralize in vitro four HIV-1 laboratory strains. Only the HIVMN variant was found to be neutralized. Compared to conventional solid phase immunoassays, the BIAcore presents several advantages for measuring the differential reactivity of peptide analogues. In view of their broadly cross-reactive potential, antibodies raised against a consensus sequence should be useful in immunodiagnosis of viral antigenic variants.

Antigenic analysis of bean pod mottle virus using linear and cyclized synthetic peptides.

The antigenic structure of the comovirus bean pod mottle virus (BPMV) was studied using synthetic peptides selected on the basis of the exposed location of certain regions of the viral protein. Three regions of domain A, four regions of domain B and two regions of domain C of BPMV coat protein were studied. Each of four regions were synthesized in the form of linear and cyclized peptides while the others were synthesized as linear peptides only. The peptides were tested for their ability to be recognized by antibodies directed against BPMV. The peptides were also used for producing rabbit antisera, which were tested for their ability to react with various BPMV antigens as well as with the linear and cyclized peptides. All the peptides were found to correspond to epitopes of BPMV coat protein. Several of the antigenic sites of BPMV located on exposed loops of the coat protein occupy positions which correspond to known epitopes in the structurally related picornaviruses. Only in some cases did cyclization sufficiently improve the level of conformational mimicry between peptides and the viral protein to allow cross-reactions between them to be observed.

Structural and dynamic studies of two antigenic loops from haemagglutinin: a relaxation matrix approach.

We have investigated the dynamics and structural behaviour of two antigenic peptides using 1H NMR. The two cyclic peptides mimic the antigenic site A of influenza haemagglutinin protein; they only differ in the way they were cyclized and in the size of their respective linkers. Homonuclear relaxation parameters extracted from a complete NOE matrix were interpreted in terms of local dynamics. A set of distance constraints was deduced from these parameters which allowed 3D models to be constructed using distance geometry. NOE back-calculation was used to check the validity of the final models. Strong variations of internal motion amplitude have been found in both peptides along their backbone. Motions with high amplitudes have been localized in the Gly-Pro-Gly sequence which forms a beta-turn in both structures.

Antigenic properties and protective capacity of a cyclic peptide corresponding to site A of influenza virus haemagglutinin.

Two cyclic peptide analogues corresponding to residues 139-146 (site A) of influenza A virus haemagglutinin (strain X31) were synthesized. The ability of these peptides to react with anti-influenza virus antibodies was found to depend on the conformation of the loop and on the orientation in which the peptide was presented to antibodies. Antibodies raised to the peptides were able to bind in ELISA with influenza virus antigen that had been allowed to dry on the microtitre plate. When OF1 mice were immunized with cyclic peptides, approximately 80% of the animals were protected against an intranasal challenge with influenza virus.

Recent advances in solid-phase peptide synthesis and preparation of antibodies to synthetic peptides.

Peptides prepared by the solid-phase peptide synthesis (SPPS) approach are used increasingly in biological research, for instance to elicit anti-peptide antibodies that will recognize the intact, cognate protein. Recent advances in SPPS are reviewed, including the use of new coupling reagents, new methods for evaluating peptide purity and new techniques of automated and multiple peptide synthesis. Methods for enhancing peptide immunogenicity are discussed such as the use of adjuvants and liposomes, and of synthetic branched polypeptides as carriers.

Synthesis of cyclic peptides on solid support. Application to analogs of hemagglutinin of influenza virus.

In order to mimic a well-known loop structure (site A) of the hemagglutinin of influenza virus, a series of cyclic peptides derived from the region 139-147 were synthesized. The lactam analogs cyclised between the N-terminus Cys 139 and the beta-carboxyl of aspartic acid 148 (small loop) or the epsilon-NH2 of lysine 148 via succinimidyl linker (large loop) were synthesized by the solid phase method. Cyclisation was directly performed on the solid support prior to final cleavage of the peptide. We describe two protection schemes which allow us to obtain different loop sizes derived from the same sequence. Eight of the analogs contained relatively large ring structures (up to 38 membered). For protection of the side chain of aspartic acid in combination with N-alpha-Fmoc protection, the cyclohexyl ester was more satisfactory than the benzyl ester with respect to imide formation. When the rate of cyclodimerisation, as a function of resin substitution, was compared to the rate of cyclic monomer formation, it was found that dimerisation was proportional to the charge of the resin. Furthermore, a comparison of the recently reported BOP reagent over the classical DIPC/HOBt method for the cyclisation reaction shows that in our case the reaction proceeded more rapidly by the BOP procedure although it gave a less pure crude product.

A branched, synthetic octapeptide of ubiquitinated histone H2A as target of autoantibodies.

Two peptides of eight (T2) and 10 (T1) residues corresponding to the branched moiety of ubiquitinated histone H2A have been synthesized and used for raising specific antibodies in rabbits. Antisera to peptide T1 reacted in ELISA with T1 and with H2A but not with ubiquitin; antisera to peptide T2 reacted with T2 but not with H2A or ubiquitin. When tested in immunoblotting, both peptide antisera reacted with ubiquitinated H2A but not with unconjugated H2A or with ubiquitin. Sera from patients with systemic lupus erythematosus (SLE) were shown previously to react with ubiquitin in ELISA and immunoblotting. When tested for their ability to react in ELISA with synthetic peptides T1 and T2, 96% of the SLE sera (diluted 1:500) that recognized ubiquitin also reacted with peptide T2. Of the SLE sera that did not react with ubiquitin, only 13% possessed antibodies able to bind peptide T2. Antibodies from seven SLE sera, purified on a T2-immunoadsorbent column, were also able to react either with H2A, and in three cases also with ubiquitin.

Specificity of antibodies raised against triacetylated histone H4.

Ten monoclonal antibodies (mAbs) were obtained by immunizing animals with triacetylated histone H4 from cuttle-fish. The fine specificity of these antibodies was studied using various populations of acetylated H4, (H3H4)2 tetramers and histone octamers as well as with acetylated and nonacetylated peptides of H4. None of these mAbs were found to recognize triacetylated H4. Only five of them bound to diacetylated, monoacetylated and nonacetylated H4. One antibody was specific for H4 associated in the form of histone octamers and did not bind to any nonacetylated or acetylated form of H4 monomers. Eight of the antibodies were specific for residues situated in the region 9-23 of H4. None of the mAbs was completely specific for acetylated forms of H4. In contrast, antisera raised in rabbits against triacetylated H4 reacted strongly with tri and diacetylated H4, weakly with monoacetylated H4 and barely or not at all with nonacetylated H4.

Comparison of different methods for localizing antigenic regions in histone H2A.

Four different variations of enzyme-linked immunosorbent assays (ELISA) were used to analyze the antigenic structure of histone H2A. Eleven natural and 10 synthetic peptides of H2A were tested for their capacity to bind antibodies raised against the complete molecule in a direct binding assay. Results were compared to those obtained in a direct test using several peptide-BSA conjugates. The capacity of peptides to inhibit the reaction between H2A antibodies and the complete H2A molecule or large fragments of it was also measured. Inhibition assays detected antigenic activity in a large number of peptides than did direct binding assays. Antisera raised against eight synthetic, unconjugated peptides all reacted with histone H2A in ELISA. Using as probes peptides of 14-21 residues, at least 11 antigenic regions could be recognized, indicating that virtually the entire H2A polypeptide chain possessed antigenic activity.