Chemically unambiguous peptide immunogen: preparation, orientation and antigenicity of purified peptide conjugated to the multiple antigen peptide system.

We described a novel and simple approach to prepare chemically unambiguous peptide immunogen using the multiple antigen peptide (MAP) approach. This approach requires the conjugation of two purified components: a chloroacetylated oligomeric lysine core matrix and a synthetic peptide containing cysteine at either the carboxyl or amino terminus. The resulting MAP is structurally unambiguous and contains a quantifiable amount of peptide antigens. Furthermore, this method also provides a flexible strategy to link a peptide antigen to the core matrix at the desirable orientation to mimic the native molecule. The carboxyl fragment 43-50 of human transforming growth factor alpha (TGF alpha) was used as a test model for this approach. Antipeptide antibodies did not recognize the "reverse immunogen" in which the peptide was attached to the MAP core matrix at a reverse orientation. To determine the specificity of the antibodies, we used two series of point-substituted TGF alpha analogs containing either alanine or the corresponding D-amino acid replacement to map the antigenic site. The alanine analogs were used to determine the contribution of the side chain while the D-amino acid analogs were used to determine the importance of backbone conformation. The antigen site was found to consist of four residues (Asp47-Leu48-Leu49-Ala50) at the distal end of the peptide-MAP conjugate. The results provide a clear explanation for the specificity of the antipeptide antibodies and their failure to recognize the "reverse immunogen" since the distal and the flexible end of the peptide-MAP construct constitutes the antigenic site. Furthermore, our results also suggests a strategy of placing the antigenic portion of a short-peptide at the distal end in the MAP approach to prepare immunogen.

[Synthetic immunogenic complexes containing a peptide from the surface protein of the foot-and-mouth disease virus]. / Sinteticheskie immunogennye kompleksy na osnove peptida poverkhnostnogo belka virusa iashchera.

Synthetic constructions containing a peptide antigenic determinant (C-terminal peptide 205-213 of the surface VP1 protein of the foot-and-mouth disease virus, O1K strain), glucosaminylmuramayl dipeptide (GMDP), and polyionic synthetic carriers were prepared. The polymerized peptide and peptide-BSA conjugates were synthesized as well. Among the constructions obtained only peptide-BSA conjugate proved to be highly immunogenic. Application of synthetic constructions to design immunogenic complexes is discussed.

Molecular bases for the construction of artificial immunogens.

The molecular and cellular mechanisms of action of synthetic polyions on immunogenesis are reviewed. The results of studies of the principal properties of polyionic immunostimulators and of the cell responses to the action of these stimulants have been used to construct artificial antigen-polyion complexes with enhanced immunogenic properties. The vaccinating properties of such macromolecular complexes, constructed with the use of bacterial or viral antigens, are analysed.

Cloning of the amino terminal nucleotides of the antigen I/II of Streptococcus sobrinus and the immune responses to the corresponding synthetic peptides.

A portion of the antigen I/II (spaA, B, P1) gene of Streptococcus sobrinus 6715, containing the coding sequence for the amino terminal 684 amino acids of the protein, was cloned in bacteriophage lambda GT10. Selection was by immunological detection using a polyclonal antiserum to the antigen I/II from Strep. mutans. From the amino acid sequence, peptides were synthesized, 15 amino acids in length, that covered the entire sequence. In total, 260 synthetic peptides were synthesized and evaluated for their immunogenicity in Balb/C mice. Thirty-nine peptides were immunogenic, without carrier, and the antisera generated were tested for their ability to bind cells of Strep. mutans and Strep. sobrinus in a solid-phase assay. Antisera corresponding to peptides from five regions on the I/II molecule bound cells of both bacterial species. These peptides were then evaluated for their ability to stimulate in vitro murine lymphocyte proliferation, after in vivo immunization with Strep. sobrinus cells. Two of the peptides were capable of stimulating proliferation, as determined by incorporation of [3H]-thymidine into murine lymph node cells. The sequences of these 5 peptides were then compared to sequences found in the antigen I/II from Strep. mutans (Kelly et al., 1989). As expected, there was considerable homology between the cross-reactive peptides synthesized and the analogous region from Strep. mutans. This homology was not usually contiguous and suggests that the antibodies bind a face of antigen I/II that is in an alpha-helical conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Quality assessment of DNA vaccines: hepatitis-B vaccine.

Vaccines manufactured following "classical" methods contain inactivated or infectious but attenuated viruses or bacteria. In some instances, the inactivated agents are purified. In other cases, the vaccines contain protein subunits or practically pure polysaccharides. It is generally accepted that the final product cannot be completely characterised and that therefore extensive "in-process" controls are necessary to prove the consistent quality of such vaccines. Control tests are carried out on the substrate, the pooled bulk vaccine and on the final containers. Vaccines produced by recombinant DNA techniques consist of pure proteins. The production is carried out by the multiplication of the "working seed" under well-defined standardised conditions followed by clarification, extraction, purification, formulation. "In-process" controls are incorporated at each step and specifications for acceptance are formulated. The biological methods used for the classical vaccines are completed by physicochemical and immunological determinations of antigen content, identity and purity for the "new generation" products. The requirements for the manufacturers are based on the documents issued by the World Health Organisation and by the national control authorities. The marketing of vaccines is based on a lot by lot release procedure, whereby each lot is tested by the manufacturer and the national control authority before use. Hepatitis-B vaccine, derived from transformed yeast cells, is the first and sole vaccine which has obtained a world-wide license. The quality assessment of this vaccine has been achieved following the requirements for the new generation of biomolecules. It is an example for future vaccines.