ALADVS · Antigenic landscape analysis of dengue virus serotypes

Antigenic landscape analysis of dengue virus serotypes

Dengue is a mosquito-borne viral disease, which has become an important international public health problem. Dengue can be caused by any of the four closely related, yet antigenically distinct serotypes of dengue viruses (DENV-1, -2, 3 and -4) that are members of family Flaviviridae (genus flavivirus). DENV causes disease ranging from mild dengue fever, to the potentially fatal dengue hemorrhagic fever / dengue shock syndrome (DHF / DSS). Antibodies elicited by DENV infection are mixture of serotype-specific, serotype-cross-reactive, flavivirus-cross-reactive antibodies, including long-lasting neutralizing antibodies to homologous DENV serotype. Presence of non-neutralising, mainly serotype-cross-reactive antibodies, enhance the infection of heterologous DENV serotype and primarily responsible for DHF / DSS. Accurate detection of anti-DENV antibodies in human sera is complicated due to the shared antigenic determinants between different flaviviruses. Despite all these issues, which are associated with the specificities of anti-DENV antibodies, our knowledge about DENV B-cell immune epitopes is very limited. In-depth information about B-cell immune epitope of each of the four DENV serotypes is required for the generation of epitope-based antigens of diagnostics and vaccine use. The major objective of this project was to identify non-cross-reactive DENV-complex and type specific epitopes, using genome fragment phage display library (GFPDL) and random peptide phage display library (RPPDL) approach, which may pave the way for the development of DENV specific antibody diagnostic test and DENV vaccine. To accomplish the goals of the project, we have first optimized the conditions for the generation of GFPDLs without any under or over representation of insert fragments from any particular part of genome. The libraries generated in monovalent phage display format were found to be functional and useful for the identification of epitopes of DENV specific monoclonal antibodies but with the polyclonal IgG / IgM antibodies from human serum, poor enrichment of clones was observed. This might be because of valence issues especially for IgM antibodies which are generally of poor affinity and this poor affinity is compensated by higher avidity. When we constructed GFPDLs of DENV-1 and DENV-2 in multivalent phage display format and panned against monoclonal and polyclonal antibodies, encouraging results were obtained. With several monoclonal antibodies, only one round of panning was required to enrich specific clones which clearly reflect that multivalent phage display format gives avidity advantage. Panning of multivalent GFPDLs on polyclonal human IgG and IgM resulted in the enrichment of immunodominant peptides / protein fragments. As it is known that envelope protein is the most immunodominant antigen of dengue virus, we have also observed maximum no. of enriched clones displaying peptides from envelope protein. In this process, we identified the epitope of one very special class of MAb (24A12) which has higher affinity to recombinant EDIII compared to native virus particle. The epitope information was used to figure out the reasons behind 24A12 special binding characteristics. In this project, we have also used RPPDLs for pannings on IgG and IgM antibodies from DENV positive human sera samples but this strategy resulted in enrichment of peptide specific for particular serum sample rather than DENV specific. Though, this strategy works for epitope mapping of monoclonal antibodies. Significant results: New vector systems for the generation of GFPDLs in monovalent and multivalent phage display format were constructed. Conditions, for the generation of GFPDLs which allow uniform distribution of inserts throughout the genome, were optimised. DENV-1 and DENV-2 GFPDLs in monovalent and multivalent phage display format were constructed and characterised on molecular and functional level. Multivalent libraries found to work better with polyclonal IgG / IgM antibodies. Using multivalent phage display libraries, we were able to identify several immunodominant epitopes of DENV-1 and -2. Epitopes of several monoclonal antibodies were also identified. Although, the funding for this project is over, we are continuing this work with intramural support. We are trying to finish all the proposed objectives which include work on remaining two DENV serotypes (DENV-3 and DENV-4) to identify immunodominant epitopes. We are also working to design chimeric antigens containing immunodominant epitopes. These chimeric antigens will be used for the specific detection of anti-dengue antibodies in human serum without any cross-reactivity problem. The availability of reliable antibody diagnostic test will have huge impact not just in clinical diagnosis of dengue infection but also in sero-surveillance / epidemiological studies and vaccine trials. The project has also built up a solid foundation for a long-term collaboration between the host and the Marie Curie fellow who is currently in the process of establishing his own research group in a high level research institute in India.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз