The use of synthetic O-antigens of Salmonella for improvement of serological diagnosis of enteric infections.

Synthetic Salmonella O-antigens of a new copolymeric type, unlike natural antigens (lipopolysaccharides), exhibit monospecificity for groupspecific factors 0 : 3, 0 : 4, and 0 : 9 of Salmonella serogroups E, B, and D, and are, by 1-2 orders of magnitude, more sensitive in double immunodiffusion and passive hemagglutination inhibition tests. The use of the synthetic antigens for detection of antibodies in patients' sera by means of passive hemagglutination increased the specificity of this test considerably, thereby improving immunodiagnosis of salmonellosis.

Quantitation of IgM antibodies to the M. leprae synthetic disaccharide can predict early bacterial multiplication in leprosy.

Quantitative enzyme-linked immunosorbent assays detecting IgM to the soluble Mycobacterium leprae crude sonicate (CD75) and the synthetic disaccharide antigen coupled to bovine serum albumin (ND-BSA) were assessed for their ability to determine early infection in families/household contacts of leprosy patients and employees of a leprosy center working in close contact with leprosy patients. Although IgM to both antigens (CD75 and ND-BSA) correlated with the bacterial index (BI) assessed histologically on skin-biopsy samples, the level of IgM antibodies to ND-BSA was a much more sensitive indicator of low bacterial loads. A 4.4-fold difference in antibody levels was observed between the mean group levels of endemic controls (N = 116) and tuberculoid leprosy patients with a BI of 0 (N = 88), increasing to sevenfold in tuberculoid leprosy patients with a BI of 1 (N = 20). Using a statistical cut off with endemic controls (mean + 2 S.D.), household/family contacts showed 30% seropositivity (N = 180) as compared to staff contacts who showed 17% seropositivity (N = 55). Percent seropositivity in family contacts was not related to the type of leprosy of the index case (lepromatous vs. tuberculoid) or the duration of treatment of the index case. Age of the individual in the family contact group had a significant influence on seropositivity. These results support the hypothesis that, in this community, factors other than the viable bacterial load of the index case, such as genetic susceptibility, may be influencing the high rate of seropositivity in family contacts. IgM ND-BSA antibodies seem to provide a good indicator of low antigenic loads and could prove to be useful in detecting subclinical infection before the onset of disease. Follow-up studies of these seropositive individuals are in progress to understand the relationship between seropositivity and the progress of clinical disease.

Regioselective syntheses of trehalose-containing trisaccharides using various glycohydrolases.

Two methods were investigated for the enzymic preparation of trehalose-containing trisaccharides. In the first, a solution of saccharides is circulated through an immobilized-glycosidase column and an activated-carbon column connected in series. In the second, two enzymes having different substrate specificities are sequentially used for condensation and subsequent specific hydrolysis. Thus, 3-O-beta-D-, 4-O-beta-D-, and 6-O-beta-D-galactopyranosyl-alpha,alpha-trehalose; 4-O-alpha-D- and 6-O-alpha-D-glucopyranosyl-alpha,alpha-trehalose; and 4-O-beta-D- and 6-O-beta-D-glucopyranosyl-alpha,alpha-trehalose were synthesized stereo- and regio-selectively.

[Synthesis of trisaccharide fragment of O-specific polysaccharides of Salmonella of serologic groups C2 and C3 and its conversion into synthetic antigen of a copolymer type]. / Sintez trisakharidnogo fragmenta O-spetsificheskikh polisakharidov Salmonella serologicheskikh grupp C2 i C3 i prevrashchenie v iskusstvennyi antigen sopolimernogo tipa.

Abequosyl-(alpha 1----3)-L-rhamnopyranosyl-(1----2)-D-mannopyranosides anomeric in the rhamnose residue have been synthesised. 2-Benzyloxycarbonylaminoethyl group used as the aglycon can be transformed into 2-acrylamidoethyl aglycon in the final stages of the syntheses. These isomeric alpha-glycosides were converted into copolymer artificial antigens, which are of interest for studying immunochemistry of factor O:8 of Salmonella O-antigens (serological groups C2 and C3).

Immunogenicity of synthetic peptides derived from the sequences of a Streptococcus mutans cell surface antigen in nonhuman primates.

The immunogenicity and antigenicity of synthetic peptides (SP) derived from the sequences of a cell surface Ag of Streptococcus mutans were investigated in macaque monkeys. Immunization with the free peptides of 11, 17, and 21 residues failed to elicit serum antibodies or T cell responses. However, immunization with the SP17 and SP21 linked to tetanus toxoid (TT) as a carrier elicited serum antibodies and proliferative responses of lymphocytes, not only to the SP but also to the native streptococcal Ag. In vivo recall of SP-TT immunized monkeys with suboptimal doses of the native streptococcal Ag resulted in a significant increase in antibodies, both to the SP and the streptococcal Ag, confirming that the SP shares antigenic epitopes with the native Ag. B and T cell epitopes were then determined and a B cell epitope was found in residues 8-13, whereas an overlapping T cell epitope was located in residues 7-15. The T cell epitope has an amino-terminal leucine and carboxy-terminal glycine and alanine added to residues 8-13 of the B cell epitope. In spite of the B and T cell epitopes being expressed in SP17 (residues 1-15), the monomer failed to induce serum antibodies without a carrier. However, immunization with a dimer of SP17 elicited both serum antibodies and proliferative responses of lymphocytes without a carrier. The results suggest that the monomeric SP17 is not immunogenic and needs to be dimerised in order to elicit antibodies and T cell responses, both to the SP and to the streptococcal Ag.

Generation of monoclonal antibodies to the specific sugar epitopes of Mycobacterium avium complex serovars.

Monoclonal antibodies have been generated to the unique distal sugar epitopes on the oligosaccharide haptens of the glycopeptidolipid antigens of clinically prominent members of the Mycobacterium avium serocomplex. Thus, antibodies are described that recognize the distal O-acetyl-alpha-L-rhamnopyranosyl residue of the specific glycopeptidolipid of M. avium serovar 1, the 4-O-acetyl-2,3-di-O-methyl-alpha-L-fucopyranose of serovar 2, the 4-O-methyl-alpha-L-rhamnopyranosyl-(1----4)-2-O-methyl-alpha-L- fucopyranosyl unit of serovar 4, the 4,6-(1'-carboxyethylidene)-3-O-methyl-beta-D-glucopyranosyl unit of serovar 8 [and the 4,6-(1'-carboxyethylidene)-beta-D-glucopyranosyl residue of serovar 21], and the 4-O-acetyl-2,3-di-O-methyl-alpha-L-fucopyranosyl-(1----4)-beta-D- glucuronopyranosyl unit of serovar 9. Epitope definition was arrived at through use of the pure, chemically defined glycopeptidolipid antigens and neoglycoproteins containing the chemically synthesized distal sugars of some select serovars. These monoclonal antibodies combined with the already published information on the structure of the antigen determinants and the tools used to arrive at these structures provide powerful means for fundamental studies on the role of these antigens in immunopathogenesis and for the precise mapping of the epidemiology of opportunistic infections caused by M. avium.

Phenolic glycolipids of Mycobacterium bovis: new structures and synthesis of a corresponding seroreactive neoglycoprotein.

The glycolipid that characterizes the majority of isolates of Mycobacterium bovis and that has come to be known as M. bovis-identifying lipid is the phenolic glycolipid mycoside B described in the literature by others. However, when mycoside B obtained from M. bovis BCG, field isolates, and infected tissues was examined in detail, it was shown to be different from that described in the literature in some important respects. In particular, the glycosyl substituent is 2-O-methyl-alpha-L-rhamnopyranose rather than 2-O-methyl-beta-D-rhamnopyranose. With this information, a seroreactive neoglycoprotein (neoantigen) containing the 2-O-methyl-alpha-L-rhamnopyranosyl substituent suitable for the serodiagnosis of bovine tuberculosis was synthesized. M. bovis also contains other minor seroreactive phenolic glycolipids, one of which is a deacylated form of mycoside B and another of which contains an alpha-L-rhamnopyranosyl unit rather than 2-O-methyl-alpha-L-rhamnopyranose.

Synthesis of p-trifluoroacetamidophenyl beta-D-glucopyranoside 4-(D-ribit-5-yl phosphate) corresponding to the Haemophilus influenzae type A capsular antigen.

The synthesis is described of the title glycoside which corresponds to the Haemophilus influenzae type a capsular antigen. The hydrogenphosphonate method was used with 3,3'-(chlorophosphonylidene)bis(2-oxo-1,3-oxazolidene) as the condensing agent.

Protective and nonprotective epitopes of chemically synthesized peptides of the NH2-terminal region of type 6 streptococcal M protein.

The protective immunogenicity of chemically synthesized copies of the NH2-terminal region of type 6 streptococcal M protein was investigated. Four overlapping peptides were synthesized by copying residues 1-20, 10-20, 12-31, and 22-31. Rabbit antisera raised against whole cells of type 6 streptococci reacted at high dilutions (1/12,800 to 1/51,200) with S-M6(1-20) and S-M6(10-20), and at low dilutions (1/100-1/800) with S-M6(12-31) and S-M6(22-31), indicating that the NH2-terminal region of type 6 M protein bears immunodominant epitopes. When covalently linked to tetanus toxoid and emulsified in complete Freund's adjuvant, the synthetic peptides S-M6(1-20), S-M6(10-20), and S-M6(12-31), but not S-M6(22-31), evoked type-specific opsonic antibodies against type 6 streptococci. Although the immune sera reacted in low dilutions by enzyme linked immunoabsorbent assay (ELISA) with the heterologous M protein polypeptides pep M5, pep M19, and pep M24, they failed to opsonize the streptococci from which these M protein polypeptides were derived. Each of the immune sera reacted in high dilution by ELISA with the respective immunizing peptides. All except those against S-M6(22-31) also reacted with pep M6. None of the immune sera reacted with human cardiac tissue by immunofluorescence or with muscle myosin by ELISA. The pattern of the inhibition of opsonization by each of the synthetic peptides of each of the immune sera indicates the presence of at least three protective epitopes in the NH2-terminal region of type 6 M protein. Our results indicate that the NH2-terminal region of type 6 M protein contains both protective and nonprotective epitopes, and chemically synthesized copies of this region lack cardiac tissue cross-reactive epitopes. These studies hold promise for the development of safe and effective vaccines against group A streptococci, especially against the strains giving rise to rheumatic fever and rheumatic heart disease.

Chemical synthesis of an octadecapeptide with the biological and immunological properties of human heat-stable Escherichia coli enterotoxin.

An eighteen-amino-acid peptide having the linear amino acid sequence of human heat-stable enterotoxin (ST) has been synthesized by solid phase peptide synthesis. The purified peptide could be obtained in yields approaching 25% after purification by size, charge, and high-performance ligand chromatography. This material was pure and identical to native ST by analytical high-performance ligand chromatography, amino acid analysis, paper electrophoresis and thin-layer chromatography. The formation of the disulfide bonds was critical for biological and immunological activity and were tentatively determined to be between cysteines 5 and 14, 6 and 10, and 9 and 17. This synthetic peptide had full immunological and biological activity when compared to native ST by enzyme-linked immunosorbent assay and the suckling mouse assay respectively.

Mercury iodide as a catalyst in oligosaccharide synthesis.

The disaccharide 4-O-alpha-D-mannopyranosyl-alpha-L-rhamnopyranose and the trisaccharide 2-O-alpha-D-galactopyranosyl-4-O-beta-D-mannopyranosyl-alpha-L-rhamno pyranose determinants, which are analogs of the repeating unit of Salmonella sero-group A, B and D, have been synthesized using mercury(II) iodide as a catalyst in the glycosylation reaction. The reducing end of the di- and the trisaccharide was substituted with a linking arm for covalent attachment to a protein carrier. Reaction of 8-ethoxycarbonyloct-1-yl 2,3-di-O-benzoyl-alpha-L-rhamnopyranoside with acetobromomannose in the presence of mercury(II) iodide gave, after deprotection, the disaccharide in 49% yield. The trisaccharide was prepared by a block synthesis in which 6-O-acetyl-4-O-allyl-2-O-(6-O-acetyl-2-O-allyl-3,4-di-O-benzoyl-alpha-D- galactopyranosyl)-3-O-benzyl-alpha-D-mannopyranosyl bromide (21) and 8-methoxycarbonyloct-1-yl 2,3-O-cyclohexylidene-alpha-L-rhamnopyranoside were condensed in the presence of mercury(II) iodide. These conditions gave the trisaccharide (26) in 26% yield. The disaccharide 21 was prepared by mercury(II) iodide catalyzed condensation of the protected galactopyranosyl bromide (15) and 4-O-allyl-1,6-anhydro-3-O-benzyl-beta- D-mannopyranose followed by acetolysis and reaction with titanium tetrabromide.

Artificial carbohydrate antigens: the synthesis of a tetrasaccharide hapten, a Shigella flexneri O-antigen repeating unit.

The 8-methoxycarbonyloctyl glycoside of the tetrasaccharide hapten, O-alpha-L-rhamnopyranosyl-(1 leads to 2)-O-alpha-L-rhamnopyranosyl-(1 leads to 3)-O-alpha-L-rhamnopyranosyl-(1 leads to 3)-2-acetamido-2-deoxy-beta-D-glucopyranoside and the trisaccharide glycoside 8-methoxycarbonyloctyl O-alpha-L-rhamnopyranosyl-(1 leads to 3)-O-alpha-L-rhamnopyranosyl-(1 leads to 3)-2-acetamido-2-deoxy-beta-D-glucopyranoside were synthesized by sequential Koenigs-Knorr reactions from monosaccharide units. The tetrasaccharide represents the complete skeletal repeating unit of Shigella flexneri serogroup Y lipopolysaccharide. Both oligosaccharide haptens are functionalized for covalent attachment to proteins, cell surfaces, and solid supports. 1H-N.m.r. evidence for the conformations of these oligosaccharides in solution is presented and shown to be consistent with predictions based on the exo-anomeric effect.