Nanobodies against the M2 protein to Influenza Virus: Preliminar results.

BARBIERI, E.S.; GARAICOECHEA, L.L.; ASENZO, G.; MARCOPPIDO, G.; PEREDA, A.; PARREÑO, V.; WIGDOROVITZ, A.

Palais Rouge, J. Salguero 1431, Buenos Aires.

Congreso; First French-Argentine Immunology Congress; 2010

Sociedad Argentina de Inmunología & French Society of Immunology.

The Influenza A vaccines currently in used are strain-specific and need to be updated every year due to antigenic drift/shift. The matrix protein M2 of influenza A is a membrane protein strongly conserved among different virus strains with an extracellular domain of 24 aminoacid residues that plays an important biological function in the virus life cycle. This domain has been the target of antiviral drugs thus we considered that it might be an excellent antigen to develop recombinant monoclonal llama-derived single-chain antibody fragments (VHH) as a pharmaceutical product to prevent influenza A infection. For this, one llama (Lama glama) was immunized with five doses of recombinant protein BLS-4M2 (Inmunova-Algenex®) emulsified with Freund adjuvant. Serum and blood samples were taken at days 0, 4 and 7 after each inoculation. The llama antibody response to M2 was monitored by ELISA and the number of specific antibody secreting cells circulating in peripheral blood was followed by ELISPOT. The ELISA antibody titers to BLS-4M2 as well as to M2 peptide were significantly increased reaching the plateau after the third inoculation (1/4096), also a very high number of M2-specific plasmocytes was obtained at the same time point (31 anti-M2 IgG ASC/5*10^5 mononuclear cells). The llama was bled two months after the last inoculation (1000 ml of blood) and the mononuclear cells were extracted from the total volume of blood to generate a phage display library. Total RNA was extracted and the cDNA was synthesized with Random Hexamer Primers. The cDNA encoding VHH was amplified by PCR using the combination of two forward primers VH1, VH6 and two reverse primers Lamb7 and Lamb8. The amplified DNA fragments were loaded in a 1,5% agarose gel and bands of a molecular weight corresponding to VHH were detected. This preliminary result demonstrates that BLS-4M2 was highly immunogenic in llama as an immunization strategy to produce a VHH library directed to M2 protein of influenza A virus.