Report 2008-2010

37
The Laboratory of Immunology and Cell Biology consists of three research
groups. In 2008-2010, the research was focussed to the following
topics: antigenic characterization of recombinant viral and bacterial
proteins, development of monoclonal antibodies (Dr. A.Žvirblienė),
regulation of gene expression by alternative splicing (Dr. A.Kanopka),
molecular epidemiology of tuberculosis (Dr. P.Stakėnas).
Antigenicity studies of recombinant viral proteins
This work was aimed at evaluating the antigenic properties of yeast-expressed
recombinant proteins that might be exploited as potential
vaccines or diagnostic tools. Using different immunochemical assays,
the antigenic structure of yeast-expressed nucleocapsid (N) proteins
of paramyxoviruses, such as human parainfluenza virus type 3 (hPIV3)
and Menangle virus has been investigated. Recombinant N proteins
were expressed at the Laboratory of Eukaryote Gene Engineering.
The yeast-expressed N proteins used in this study were self-assembled
A
into nucleocapsid-like structures (NLPs) similar to that of native virus.
To identify B-cell epitopes, we have employed monoclonal antibodies
(MAbs) raised against recombinant N proteins as well as human sera
from virus-infected individuals. The localization of B-cell epitopes was
studied using recombinant overlapping N protein fragments, PepScan
analysis and competitive ELISAs. The majority of MAb epitopes were
mapped within the C-termini of N proteins. Cross-inhibition studies
with human sera demonstrated similar localization of B cell epitopes
recognized by serum antibodies from naturally infected individuals,
which revealed a clear antigenic similarity between recombinant and
virus-derived N proteins. Our data suggest that different paramyxoviruses
may contain highly conserved surface-exposed structures
within the C-termini of N proteins that are well accessible to B cells
and elicit antibody response. These findings may have important implications
for the design of new vaccines and diagnostic reagents (Zvirbliene
et al., Viral Immunol. 2009, Zvirbliene et al., Arch Virol. 2010).
B
Figure 1. Immunochemical detection of virus-infected cells using monoclonal antibodies raised against recombinant N proteins of hPIV3 (A) and Menangle virus (B).
Functional and immunological characterization of recombinant
vaginolysin
Vaginolysin (VLY) is the main virulence factor released by Gardnerella
vaginalis. VLY is a protein toxin that acts as a hemolysin. It belongs to
the group of cholesterol-dependent cytolysins (CDCs), a large family
of related pore-forming toxins found in five different genera of Grampositive
bacteria. Generation of MAbs against bacterial cytolysins may
promote elucidation of the relationship between their structure and
mode of action.
In the current study, we have developed and characterized a panel of
MAbs against recombinant VLY and demonstrated that several MAbs
neutralize VLY in the in vitro hemolytic assay. By using a series of recombinant
proteins we have studied the specificity of the MAbs and
mapped the epitope recognized by the most potent neutralizing MAb.
The epitope for this MAb was localized near the N-terminus of VLY
and included the conserved motif (VAARMQYD, aa 189-196) supposed
to be involved in VLY oligomerization. Furthermore, we have
employed the MAbs in a sandwich ELISA for VLY quantitation. In conclusion,
the MAbs described in the current study may be useful for
structural and functional studies of VLY as well as immunodetection
of VLY in biological specimens (Zvirbliene et al., Toxicon 2010,
PCT/LT2009/000005).
This work was supported by the Lithuanian State Science and Studies
Foundation/Lithuanian Science Council (grant No. N-17)
Dr. M.Plečkaitytė and Master student J.Alesiūtė investigate immunochemical
properties of vaginolysin
Figure 2. Schematic representation of VLY domains (D1-D4). Dashed area indicates VLY region recognized by the neutralizing monoclonal antibody.