Report 2008-2010

Master student V.Žilaitytė, Dr. A.Žvirbliene, junior scientist I.Šėžaitė, PhD student
I.Kučinskaitė-Kodzė, junior scientist R.Lasickiene develop new hybridoma cell lines
Junior scientist E.Jakubauskienė and PhD student I.Pečiulienė investigate HIF-3a
expression
Generation of monoclonal antibodies of desired specificity using
chimeric virus-like particles
Protein engineering provides an opportunity to generate new immunogens
with desired features. Viral structural proteins with their intrinsic
capacity to self–assemble to highly-organized virus-like particles
(VLPs) have been shown to possess high immunogenicity and have
been exploited as potential vaccines. Previous studies demonstrated
that insertions/fusions of foreign protein segments at certain sites of
VLP carriers did not influence protein folding and assembly of chimeric
VLPs.
In the Laboratory of Eukaryote Gene Engineering, chimeric VLPs representing
major capsid protein VP1 of hamster polyomavirus (HaPyV)
with inserted foreign sequences at certain surface-exposed regions
were expressed in yeast S.cerevisiae. These chimeric HaPyV-VP1 VLPs
have been shown to induce a strong antibody response against the
inserts (Gedvilaite et al., 2004, Zvirbliene et al., 2006). Chimeric VLPs
efficiently activate both B cells recognizing the surface-located epitopes
and T helper cells providing the necessary signals for Ig class
switching and affinity maturation.
We have employed chimeric VLPs harbouring foreign sequences of
different size and origin to generate insert-specific MAbs. The length
of inserts ranged from 6 to 280 amino acids. It was demonstrated that
chimeric VLPs efficiently stimulated the production of IgG antibodies
specific for the sequences/epitopes presented at surface-exposed regions.
This approach was successfully used to generate MAbs against
non-immunogenic protein sequences, such as tumor-associated epitopes
(Dorn et al., Viral Immunol, 2008). Moreover, it was demonstrated
that the insert-specific antibodies recognized native full-length
proteins, which suggested the correct folding of the sequences displayed
on VLPs. Our data confirm that the insertion of non-immunogenic
epitopes into VLPs significantly increases their ability to
induce a strong B cell response. Thus, chimeric VLPs represent highly
efficient immunogens for hybridoma technology and provide a promising
alternative to chemical coupling of synthetic peptides to carrier
proteins.
This work was supported by UAB Fermentas (Contract No. 1224).
Regulation of hypoxia-inducible factor HIF-3α expression via the
alternative pre-mRNA splicing mechanism.
Recent genome-wide analyses of alternative splicing indicate that up
to 70% of human genes may have alternative splice forms, suggesting
that alternative splicing together with various posttranslational modifications
plays a major role in the production of proteome complexity.
Changes in splice-site selection have been observed in various types of
cancer and may affect genes implicated in tumor progression and in
susceptibility to cancer. This may lead to altered efficiency of splice-site
recognition, resulting in overexpression or down-regulation of certain
splice variants, a switch in splice-site usage, or failure to recognize splice
sites correctly, resulting in cancer-specific splice forms. At least in some
cases, changes in splicing have been shown to play a functionally significant
role in tumorigenesis, either by inactivating tumor suppressors
or by gain of function of proteins promoting tumor development.
Thus, the identification of cancer specific splice forms provides a novel
source for the discovery of diagnostic or prognostic biomarkers and
tumor antigens suitable as targets for therapeutic intervention.
Figure 3. Schematic representation of HIFα subunit mRNA variants detected in
the cell.
35 th anniversary
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