Dissertation for Scientific Degree of Dr.biol. (in Molecular Biology)
Guna Mezule
The linkage between location and length of inserted sequences and
their antigenicity and immunogenicity in chimeric proteins.
University of Latvia, Riga, 1997
SUMMARY
The immunological properties of proteins are determined by their primary
structure and conformation. Therefore peptides are not able to mimic the behavior
of native proteins, even if their amino acid sequence is identical, their geometry
may not correspond to that of the protein fragment. Achievements in the field of
genetic engineering gave a possibility to create new chimeric recombinant
proteins, where in the capsids, which serve as carrier moieties, is possible to
clone epitopes from foreign proteins. Such recombinant molecules are much more
suitable for studying of protein immunological properties on the molecular level.
The objects of this study were new chimeric recombinant proteins,
where truncated hepatitis B virus core (HBcA) protein was used as a carrier
moiety and immunodominant epitopes from foreign proteins were inserted at
position 78 or 144 within HBcA molecule. We used foreign viral sequences from:
1) HIV-1 gp120 variable loop (V3);
2) HIV-1 p24;
as well as non-viral sequence from bovine inhibin a-chain.
The general aim of this study was to elucidate the positioning of the inserted
foreign sequences in such chimeric proteins, whether on the surface or internal,
and to find linkage between location and length of inserted sequences and their
antigenicity and immunogenicity in chimeric proteins.
We fuond that region 160-192 of p24 within HBc-p24 is exposed on the surface
of chimeric capsids, whereas the region 133-157 is buried. In the case of V3 loop
from gp120 insertion, we observed the full superficial accessibility of inserted V3
region for specific anti-V3 antibodies in all proteins studied. It was proved that
immunogenicity of inserted foreign sequences within HBc particles is dependent
strongly on their location on the particles, superficial or internal, and their length.
Insertions induce stronger specific antibody response if they are placed within the
major immunodominant region of HBcA molecule at aa 78, and when they are
longer.
During this study we concluded that it is possible to decrease strongly
antigenicity and immunogenicity of carrier moiety - HBc protein, by dissection of
major immunodominant region with foreign insertion and by truncation of its Cterminus.
The present work is described in 3 papers and 1 patent application and
presented in 5 international conferences.
LIST OF ORIGINAL PAPERS
This thesis is based on the following papers, which are referred to by their
Roman numerals:
I Grene, E., Mezule. G.. Borisova, G., Pumpens, P., Bentwich, Z., & Amon, R.
(1997). Relationship between antigenicity and immunogenicity of chimeric
hepatitis B virus core particles carrying HIV type 1 epitopes. AIDS Research
and Human Retroviruses 13, 41-50.
II Borisova, G., Borschukova-Wanst, 0., Mezule. G.. Skrastina, D., Petrovskis, I.,
Dislers, A., Pumpens, P. & Grens, E. (1996). Spatial structure and insertion
capacity of immunodominant region of hepatitis B core antigen. Intervirology
39, 16-22.
Ill Borisova, G., Borschukova, 0., Skrastina, D., Mezule. G.. Disler, A., Petrovskis
I., OseV., Gusars, I., Pumpens, P. & Grens, E. (1997). Display vectors. I.
Hepatitis B core particle as a display moiety. Proc. Latv. Acad. Sci. 51, 1-7.
Borisova, G., Borschukova, 0., Mezule. G.. Petrovskis, I., Pumpen, P., Skrastina,
D. & Ireland, J. J. (1997). Inhibin-HBc fusion protein. U.S. Patent Application.