Development of human monoclonal antibodies to influenza virus from Fab fragment library

• Grant number: 22/00228-0
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: March 01, 2022
• End date: February 28, 2026
• Field of knowledge: Biological Sciences - Immunology - Applied Immunology
• Principal Investigator: Ana Maria Moro
• Grantee: Kenneth Gabriel Mota
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Associated research grant: 20/07040-1 - Center of Research and Development in Immunobiologicals, AP.NPOP

Abstract

The viruses that cause seasonal influenza infect between 5-15% of the world's population annually, causing around 650,000 deaths/year. Every year there is a recurrence of seasonal epidemics caused by Influenza; epidemiological data show that in Brazil 29,978 cases of SARS (Severe Acute Respiratory Syndrome) occurred in 2019 and, of these, 4,911 cases (16.4%) were due to seasonal Influenza. Vaccination needs annual update to follow the viral evolution. Patients at risk of severe cases could benefit from a specific treatment based on neutralizing monoclonal antibodies (mAbs). MAbs with high affinity for the target can neutralize the virus in the host organism, alleviating severe symptoms of the disease. Human mAbs can be obtained by the phage display technique, which consists in the Fab presentation by the phages present in a library and the selection of mAbs by binding to an antigen. The first human mAb developed by this technique and approved by the FDA (Food and Drug Administration) was Adalimumab (Humira®), used as a human TNF-± inhibitor. The aim of the present study is to select by phage display, human Fab fragments with broad neutralizing potential for different influenza virus strains. The influenza antigens in this study will come from the Butantan Institute strain bank. A naïve Fab library constructed in the laboratory will be used for the screening. The library will be transformed into E. coli XL1-Blue and amplified, later will be infected with helper phage to obtain phages presenting Fab. The library will be enriched by successive rounds of panning by binding to the antigen. Phage ELISA assays will be performed for Fab selection from the enriched library. The promising Fabs selected from the previous steps will be expressed and purified, for their characterization and for neutralization tests. The obtained Fabs will be tested for their affinity to the antigen by ELISA assay and subsequently the best Fab clones will be submitted to Western blot assay for their characterization. Affinity assays of the Fab to the antigen will be done by surface plasmon resonance. Then Fabs selected from the previous steps will be evaluated for viral neutralization by hemagglutination inhibition assays (HIC).