Molecular presenting system

Details Molecular presenting system Molecular presenting system

1997-03-31

1999-08-03

Ketter, James

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

435 691, 4353201, 435472, 530300, 5303894, 536 2372, G01N 3353, C12P 2100, C07K 1600, C07H 2104

Patent

active

059324260

DESCRIPTION:

BRIEF SUMMARY
TECHNICAL FIELD

The invention concerns a molecular presentation system in which viral proteins are being used as carriers for heterologous amino acid sequences.


BACKGROUND OF THE INVENTION

The possibility to identify and synthesize amino acid sequences from viral proteins, which are able to generate a protective immune response in animals, has stimulated the development of synthetic vaccines. Although it has already been shown that synthetic peptides in some cases can induce a good immune response, it has turned out that in general they were weak immunogens unless coupled to strongly immunogenic carrier molecules. They were frequently unable to induce protective immunity in vaccinated animals. Attempts to increase the immunogenicity of these antigens for use as vaccine have lead to the development of a series of antigen presentation systems. Many of these are designed to present the antigen as a polyvalent, particulate structure. The development of particulate vector systems for immunogenic epitopes provides a powerful approach for the presentation of antigens. Various systems were used to present foreign
It has been demonstrated that the immunogenicity of a peptide depends on its sequence as well as on the way it is presented to the immune system. By using a human rhino virus capsid sequence as a heterologous peptide and the particles of HBcAg as a carrier, it was shown that the internal location of the foreign sequence increases the immunogenicity of the epitope by 10 to 50 fold when compared to the amino terminus location antibody (mAb)) was greatly enhanced by placing the foreign peptide in that position in the carrier. Furthermore, both constructs presented the epitopes considerably more efficiently to the mAbs than the free peptides. This was also the case when specific HIV-1 epitopes (the V3 loop) were of a given epitope can be influenced by its conformation it was of great interest to have a carrier system with multiple entry sites conferring many possible conformations. This would increase the possibility of finding a conformation closer to the native one for a given sequence. In spite of the fact that, as mentioned above, various particulate systems have been developed for the presentation of epitopes, they were all based on the foreign epitope being inserted mainly in one position. This was partly due to lack of knowledge about the 3-D structure of the carrier particle.


SUMMARY OF THE INVENTION

With reference to the above, a new presentation system has been developed, characterized by the fact that the carrier protein is derived from small insect viruses, Flock House virus (FHV), with a known 3-D structure and amino acid sequence. Heterologous amino acid sequences, for example epitopes, are inserted into the outwards directed loops of the viral capsid protein. This carrier presents multiple possibilities for a conformationally suitable location of epitopes. Above all, the carrier system is characterized by the fact that the recombinant protein, or the virus like particles, are obtained from procaryotic or eucaryotic cells through the expression of the protein encoded by the appropriately modified RNA-2 gene of the FHV capsid protein.


BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a crystallographic representation of the outward directed loops of the FHV capsid protein precursor with 3 Angstrom resolution showing the positions of insertions of the foreign genes (L1, L2, L3, I1, I2 and I3).
FIGS. 2A-2C depict the DNA and amino acid sequences of FHV RNA-2 with the individual loops (L1, L2, L3, I1, I2 and I3) shown in bold type.
FIGS. 3A-3F depict the full restriction map of the DNA sequence of FHV RNA-2.
FIGS. 4A-4B depict the number of restriction endonuclease usage sites in the FHV RNA-2.
FIGS. 5A-5D depict all of the restriction endonuclease sites in the FHV RNA-2.
FIG. 6 is a graphic representation of the unique endonuclease sites in the FHV RNA-2.
FIG. 7 depicts a stereo diagram of the FHV RNA-2 protein showing the positions in which HIV-1 specific sequences are inserted.
FIG.

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