Founded in 1975 as the all Union Research Institute of Applied Enzimology, currently,  the Institute of Biotechnology is mainly involved in research and training in the fields of biotechnology and molecular biology, including research and development of recombinant biomedical proteins, genetic and molecular studies of restriction modification phenomenon, developing of viruses diagnostics, epigenetic study of small RNA, drug design and synthesis, bioinformatics.

Antigenicity studies of recombinant viral proteins and development of monoclonal antibodies

Spausdinti

Antigenic characterisation of yeast-expressed viral proteins (Dr. A.Zvirbliene)


This work was performed in collaboration with the Laboratory of Eukaryote Gene Engineering. It 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 measles nucleocapsid (N) protein and lyssavirus N proteins was investigated. These recombinant antigens were expressed in the Laboratory of Eukaryote Gene Engineering.
The yeast-expressed measles N protein used in this study was self-assembled into nucleocapsid-like structures similar to that of native virus. To identify B-cell epitopes in measles N protein, we have employed monoclonal and polyclonal antibodies raised against recombinant N protein as well as human sera from measles-positive individuals. The localization of B-cell epitopes was studied using recombinant overlapping N protein fragments, PepScan analysis and competitive ELISA. The majority of monoclonal antibody epitopes were mapped within the C-terminus of N protein. Cross-inhibition studies with human sera demonstrated similar localization of B cell epitopes recognized by serum antibodies from naturally infected individuals, which reveals a clear antigenic similarity between recombinant and measles virus-derived N protein. The results of the current study are in agreement with previous structural studies of measles N protein showing the accessibility of the C-terminal domain on the surface of viral nucleocapsids. These findings may have important implications for the design of new recombinant measles vaccines and diagnostic reagents (Zvirbliene et al., 2007).
In Europe, three genotypes of the genus Lyssavirus, family Rhabdoviridae, are present: classical rabies virus (RABV, genotype 1), European bat lyssavirus type 1 (EBLV-1, genotype 5) and European bat lyssavirus type 2 (EBLV-2, genotype 6). The N proteins of RABV, EBLV-1 and EBLV-2 were expressed in yeast Saccharomyces cerevisiae and purified by density-gradient centrifugation. As demonstrated by electron microscopy, the purified N proteins were self-assembled to nucleocapsid-like structures. The antigenic structure of the N proteins was investigated for their reactivity with monoclonal antibodies (mAbs) directed against different lyssaviruses. The reactivity pattern of each mAb was virtually identical between immunofluorescence assay with virus-infected cells, and ELISA and dot blot assay using the corresponding recombinant N proteins. These observations lead us to conclude that yeast-expressed lyssavirus N proteins share antigenic properties with naturally expressed virus protein. These recombinant proteins have the potential for use as cost-effective antigenic components of serological assays for lyssaviruses. This could find application in the assessment of the response to rabies vaccination and a virus-specific screening antigen for the measurement of seroprevalence for active surveillance of EBLVs in European bats (Kucinskaite et al., 2007).


Generation of monoclonal antibodies of desired specificity using chimeric virus-like particles (Dr. A.Zvirbliene)


The production of monoclonal antibodies (mAbs) directed against selected epitopes or peptides has important implications for functional and clinical studies. Such antibodies have become indispensable tools in biochemistry and molecular biology, including microarray-based proteomics. For the generation of anti-epitope antibodies, synthetic peptides are commonly used. Because of their size, peptides are usually not immunogenic and have therefore to be coupled to larger carrier proteins. An efficient immune response to the desired peptide can be achieved with a careful selection of the carrier by which the peptide can be displayed to the immune system. Synthetic peptides when coupled to carrier proteins such as bovine serum albumin, ovalbumin or keyhole limpet hemocyanin, usually elicit a strong humoral immune response. However, many factors influence the immunogenicity of the peptide, such as method of coupling, length of the sequence, its hydrophilicity, accessibility, mobility and protrusion. The problem that is encountered when preparing anti-peptide antibodies is whether the peptide sequence is displayed on the surface of the peptide-carrier conjugate to be accessible to B cells. It is well known that the antigenic parts of a protein surface are located predominantly in loops and/or protruding regions.
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 Saccharomyces cerevisiae. These chimeric HaPyV-VP1 VLPs have been shown to induce in mice a strong antibody response against the inserts (Gedvilaite et al., 2000, 2004). Thus, the chimeric VLPs meet the requirements for a strong immunogen being able to 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. Moreover, it was demonstrated that the insert-specific mAbs 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 efficient immunogens for hybridoma technology and provide a promising alternative to chemical coupling of synthetic peptides to carrier proteins (Zvirbliene et al., 2006).
Based on these investigations, the patent application was filed (PCT/EP2006/003420, WO2006/108658). The licensing agreement with UAB Fermentas was signed (20.12.2006).
This work was supported by the Lithuanian State Science and Studies Foundation (grants No. B-16/2006, G-06/07) and UAB Fermentas (grant No. 11/07).

Partners:
Dr. R.Ulrich, Friedrich-Loeffler Institute, Greifswald-Insel Riems, Germany
Dr. V.Gorboulev, Wurzburg University, Wurzburg, Germany
Dr. Z.Gudleviciene, Vilnius University, Institute of Oncology
Dr. I.Jonauskiene, Vilnius University, Institute of Immunology

Grants:
Lithuanian State Science and Studies Foundation
EU Structural funds

Contracts:
UAB Fermentas (Vilnius, Lithuania)
Abcam Ltd (Cambridge, UK)
Santa Cruz Biotechnology (USA)
Customs laboratory (Vilnius, Lithuania)