Synthetic peptides for Plasmodium vivax malaria sero-epidemiology. Application of Fmoc-polyamide and displacement chromatography.

The immunodominant epitope of Plasmodium vivax, one of the major causative agents of malaria in man, consists of the tandem repetitions of a nonapeptide sequence, AspArgAlaAsp/AlaGlyGlnProAlaGly, with Asp (variant d) or Ala (variant a), in the fourth position. Synthetic peptides corresponding to the P. vivax epitope, containing a different number of nonapeptide sequences, were prepared by solid-phase synthesis according to the Fmoc-polyamide method. Three peptides, containing 1, 2, and 4 copies of the d variant, were assembled on the gel polymer; none of these peptides, however, was suitable for P. vivax sero-epidemiology. A 45-peptide containing both the d and a variants, ddaad, was prepared by continuous-flow Fmoc-polyamide (flow-polyamide). Among the cleavage procedures evaluated for the removal of the five Mtr groups only TFMSA/TFA/1,2-ethanedithiol (1:89:10 by vol) brought deblocking to completion; a substantial level of impurities originated, however, from these procedures. The product was purified by reversed-phase displacement chromatography, a technique only recently applied to peptides, which shows distinct advantages over conventional, linear elution chromatography. In a single experiment, 107 mg of the crude mixture were loaded onto an analytical column (250 x 4 mm), obtaining in purified form 85% of the desired material present in the sample. An ELISA test base on the ddaad peptide was developed and is being applied to the sero-epidemiology of P. vivax malaria.

Polarity of immunogens: implications for vaccine design.

Peptide constructs have been engineered consisting of amino acid sequence determinant recognized by T cells (TD) co-linearly linked to haptenic peptides. It was found that high anti-hapten antibody titers were induced after immunization with those constructs which had the TD sequence in the N-terminal position with respect to the hapten. Low or zero titers were elicited when the TD was in C-terminal position. Also, a high anti-hapten antibody titer corresponded to a low or zero anti-TD antibody titer and vice versa. These results suggest that immunogens are polar and stress the relevance of searching the more adequate position of the TD within a peptide construct when designing immunogens or synthetic peptide vaccines.

Protective anti-sporozoite antibodies induced by a chemically defined synthetic vaccine.

Chemically defined synthetic polymers, known as multiple antigen peptide systems (MAPs) represent an effective and novel approach for engineering peptide-based vaccines. Ten different mono and diepitope MAP models, containing different arrangements and stoichiometry of functional B and/or T helper epitopes from the circumsporozoite protein of Plasmodium berghei were used to immunize mice. High titers of antibody and protective immunity against sporozoite challenge were elicited by MAPs containing T and B epitopes arranged in tandem and in equimolar amounts. These results indicate that MAPs may serve as a basis for developing subunit vaccines to induce high levels of antibodies against sporozoites.

Epitopes of the mycobacterial heat shock protein 65 for human T cells comprise different structures.

T cell recognition of foreign antigens is a result of a ternary complex between T cell receptor, nominal peptide and major histocompatibility complex molecule. It has been proposed that the nominal peptide, which is presented by accessory cells to T cells, has a characteristic structure which can be predicted on the basis of physicochemical criteria. To further study this aspect, we stimulated T cells from normal human blood donors with synthetic peptides (each of approximately 15 amino acids in length) from the heat shock protein 65 of Mycobacterium tuberculosis-M. bovis. We found that while the characterization of certain epitopes follows commonly used predictions, other epitopes cannot be predicted by known methods.

[Synthesis and immunological properties of the peptide fragment of the hepatitis B virus envelope preS-protein]. / Sintez i immunologicheskie svoistva peptidnogo fragmenta preS-oblasti belka obolochki virusa gepatita B.

We have synthesized the 24-41 fragment of the preS region of the hepatitis B (subtype ayw) envelope. The peptide was prepared by the solid phase synthesis on perfluoropoly-ethylene polymer grafted with polystyrene. The peptide chain was elongated from C-terminus using pentafluorophenyl- and p-nitrophenyl esters of Boc-amino acids. The peptide was cleaved off the solid phase by HBr in CF3COOH, purified by gel filtration, and, after conjugation with serum albumin (BSA), inoculated into mice. The resultant antibodies were shown to react with the peptide. The blood sera from patients with acute hepatitis B reacted with the peptide-BSA conjugates in the immunoenzymic solid phase assay.

Synthesis and biophysical characterization of engineered topographic immunogenic determinants with alpha alpha topology.

Model peptides with predetermined secondary, tertiary, and quaternary conformation have been successfully designed, synthesized, and characterized in an attempt to mimic the three-dimensional structure of an antigenic determinant. This work is a continuing effort to map the antigenic structure of the protein antigen lactate dehydrogenase C4 (LDH-C4) to develop a contraceptive vaccine. A putative topographic determinant with alpha alpha topology which associates into four-helix bundles was designed on the basis of the framework model of protein folding. An idealized amphiphilic 18-residue sequence (alpha 1) and a 40-residue alpha alpha fold (alpha 3) have been shown to form stable 4-helix structures in solution with a free energy of association on the order of -20.8 kcal/mol (tetramerization of alpha 1) and -7.8 kcal/mol (dimerization of alpha 3). Both alpha 1 and alpha 3 form stable monolayers at the air-water interface. The CD spectra of Langmuir-Blodgett monolayers are characteristically alpha-helical. Both CD and FTIR spectroscopic studies reval a high degree of secondary structure. The SAXS data strongly suggest that the helices are arranged in a four-helix bundle since the radius of gyration of 17.2 A and the vector distribution function are indicative of a prolate ellipsoid of axial dimensions and molecular weight appropriate for the four-helix bundle. The major contribution to the formation and stabilization of alpha 1 and alpha 3 is believed to be hydrophobic interaction between the amphiphilic alpha-helices. The displayed heptad repeat, helix dipole, ion pairs, and the loop sequence may have also contributed to the overall stability and antiparallel packing of the helices. A detailed structural analysis of a relevant topographic immunogenic determinant will elucidate the nature of antigen-antibody interactions as well as provide insight into protein folding intermediates.

Lymphocyte responses to peptide M and to retinal S-antigen in uveitis patients.

A synthetic amino acid, peptide M18 (18 amino acids in length which correspond to the amino acid positions 303-322 of bovine S-antigen: DTNLASSTIIKEGIDKTV), is capable of inducing experimental autoimmune uveitis in monkeys and Hartley guinea pigs as well as Lewis rats. Recently, the amino acid sequence of human S-antigen corresponding to peptide M was found to be virtually identical to that of bovine S-antigen. To investigate the role of the epitope corresponding to peptide M in human uveitis, 21 patients with uveitis were tested for proliferation of peripheral mononuclear cells against peptide M18 and bovine S-antigen. Six of the 21 patients responded to S-antigen and 4 of these 6 patients responded to peptide M18. These findings show that peptide M18 is immunogenic in man and provide additional support to the hypothesis that the amino acid sequence which corresponds to peptide M might be involved in uveitic conditions in man.

Antibodies as probes of the structure and function of the nicotinic acetylcholine receptor.

Structural and functional studies of the nicotinic acetylcholine receptor from Torpedo marmorata are discussed as examples for the distinct differences in properties of antibodies raised against a native antigen as compared to antibodies raised against short or long synthetic peptides.

A serine protease (CCP1) is sequestered in the cytoplasmic granules of cytotoxic T lymphocytes.

Based upon the use of a new predictive algorithm, three peptides were synthesized that correspond to likely antigenic sites of the cytotoxic T cell-specific protease cytotoxic cell protein 1 (CCP1). Antibodies raised against these peptides, under reducing conditions, bound to a single protein of m.w. 29,000 found in two actively cytotoxic T cells. This protein was absent from a "cytotoxic" T cell line that had lost its cytolytic properties. By using immunocytologic techniques, the protein was localized within cytoplasmic granules. In addition, granules purified from the cytoplasm of cytotoxic T cells were shown, by Western blot analysis, to contain the protein. The molecule detected by the antibodies behaves, upon reduction, similarly to a diisopropylfluorophosphate-binding protein. Thus, here we provide evidence that CCP1, a protein whose expression correlates with cytotoxicity, is contained within organelles which have been implicated in killing. These findings strongly suggest that CCP1 plays a key role in T cell-mediated target cell lysis.

Molecular cloning and chemical synthesis of a region of platelet glycoprotein IIb involved in adhesive function.

Membrane glycoprotein (GP) IIb-IIIa is a component of a platelet adhesive protein receptor. A region of the heavy chain of GPIIb, defined by the monoclonal antibody PMI-1, is involved in adhesion receptor function. We have localized and chemically synthesized this region of GPIIb. A cDNA clone that directs the synthesis of a fusion protein reactive with the PMI-1 antibody was isolated from a phage lambda gt11 expression library constructed with mRNA from an erythroleukemia (HEL) cell line. The deduced amino acid sequence of this clone indicates that it spans the light-heavy chain junction of GPIIb and contains a portion of the carboxyl terminus of the heavy chain and the amino terminus of the light chain. The PMI-1 epitope was found to be contained within a 9-kDa staphylococcal V8 protease fragment of GPIIb, and such a fragment was predicted within the putative heavy-chain sequence. A computerized antigen prediction program identified a single sequence with a high probability of containing a continuous epitope. A synthetic 17-residue peptide containing this sequence binds PMI-1 and inhibits PMI-1 binding to GPIIb-IIIa. The peptide-antibody complex has an approximate Kd of 1.2 microM, which compares to a Kd of 0.95 microM for PMI-1 binding to GPIIb. The region containing the PMI-1 epitope shows no similarity to corresponding regions of two other adhesion receptors, indicating that this portion of GPIIb may function in activities unique to the platelet receptor.

T cell recognition of a tumor-associated glycoprotein and its synthetic carbohydrate epitopes: stimulation of anticancer T cell immunity in vivo.

The Thomsen, Friedenreich (TF) and Tn carbohydrate antigens are expressed on the vast majority of human adenocarcinomas and are associated with aggressive behavior of certain tumors. TF and Tn antigens are also expressed on certain murine cancer cell lines including TA3-Ha, a highly lethal, transplantable mammary adenocarcinoma. TF and Tn cancer-associated carbohydrate haptens were synthesized, conjugated to protein carriers and used to demonstrate that delayed-type hypersensitivity (DTH) effector T cells can specifically recognize and respond to carbohydrate determinants on the TA3-Ha tumor-associated glycoprotein, epiglycanin. The effector cells were shown to have the helper DTH phenotype (Lyt1+, Lyt2-, Thy1+) and it was demonstrated that they respond to specific carbohydrate determinants in an MHC-restricted fashion. These experiments provide the rationale for the use of synthetic tumor-associated glycoconjugates (S-TAGs) to stimulate anticancer T cell immunity. In support of this hypothesis, it was shown that preimmunization with the appropriate S-TAGs could provide a degree of protection against a subsequent tumor transplant and that antitumor effector Lyt1+, Lyt2- T cells could be generated in vitro using the appropriate S-TAGs as antigens.

Chemical synthesis of the desialylated human Cad-anti-genic determinant.

Benzyl 2-azido-2-deoxy-beta-D-galactopyranoside was converted into benzyl 2-azido-4,6-O-benzyl-2-deoxy-beta-D-galactopyranoside via benzylidenation, p-methoxybenzylation, acid hydrolysis, benzylation, and selective oxidation. Condensation of 1,2,3,4,6-penta-O-acetyl-beta-D-galactopyranose with benzyl 2-azido-4,6-di-O-benzyl-2-deoxy-beta-D-galactopyranoside in the presence of trimethylsilyl triflate gave crystalline benzyl 2-azido-4,6-di-O-benzyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-ga lactopyranosyl)-beta-D-galactopyranoside (76%), which was converted into benzyl 2-azido-4,6-di-O-benzyl-2-deoxy-3-O-(2,6-di-O-benzyl-beta-D-galactopy ranosyl)-beta-D-galactopyranoside and condensed with 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha-D-galactopyranosyl bromide in the presence of silver silicate on alumina and molecular sieve 4 A to give 61% of benzyl O-(3,4,6-tri-O-acetyl-2-azido-2-deoxy-beta-D-galactopyranosyl)-(1----4)- O-(2,6-di- O-benzyl-beta-D-galactopyranosyl)-(1----3)-2-azido-4,6-di-O-benzyl-2-deo xy- beta-D-galactopyranoside. Reduction with sodium borohydride followed by N-acetylation, O-deacetylation, and catalytic hydrogenolysis then gave O-(2-acetamido-2-deoxy-beta-D-galactopyranosyl)-(1----4)-O-beta-D-gal actopyranosyl-(1----3)-2-acetamido-2-deoxy-D-galactopyranose, the desialylated human Cad-antigenic determinant.

Highly immunogenic artificial complexes based on synthetic immunopotentiating polyions (artificial antigens and vaccines).

An interpretive review of the theoretical and experimental data on the immunogenetic concept that "there are no strong and weak antigens, there are high and low responder genotypes," developed and upheld by the authors is presented. To achieve phenotypic correction (finding ways of turning genetically low responder individuals into high responder ones) the authors have developed complex antigens--artificial macromolecular complexes containing both the required antigenic determinants and adjuvant structures. Synthetic nonnatural carbohydrates and heterochain polyions with controlled structure (PAA, PVP, polyconidine quarternary salts) were shown to act as immunopotentiators, substituting the helper signal of T-cells. The authors' data confirm this immunogenetic principle for developing highly immunogenic preparations, prototypes of future vaccines.