Structural and biophysical investigations toward engineering a protein subunit vaccine for human metapneumovirus

Human metapneumovirus (hMPV) is an enveloped negative-sense RNA virus in the family Pneumoviridae. Although identified in 2001, it has been circulating for at least 60 years and is now recognized as a leading cause of acute lower respiratory tract infections in young children and the elderly. It is a ubiquitous pathogen that infects nearly all children by the age of 5, but children younger than 12 months old are at an increased risk of developing more severe disease such as bronchiolitis and pneumonia. Despite this early viral exposure, reinfections are common throughout life. In subsequent infections, the symptoms experienced by healthy adults will typically consist of mild upper respiratory distress. However, hMPV infections can lead to higher rates of morbidity and mortality among the elderly, especially those with comorbidities. Currently, no therapeutics or vaccines have been approved by regulatory agencies for the treatment of human metapneumovirus. Therefore, the work in this dissertation aims to provide not only potential vaccine and therapeutic candidates, but also reagents that may be beneficial to the human metapneumovirus field. The first chapter sets out to establish the significance of acute lower respiratory tract infections and their contribution to the global burden of disease. A brief history of hMPV is then covered before delving into a general description of the virus’ molecular biology. The second chapter explores the use of structure-based vaccine antigen design to engineer a human metapneumovirus protein subunit vaccine antigen. The previously described prefusion hMPV F structure is used as a guide to design mutations that are expected to decrease the propensity of the protein to undergo conformational change. Biophysical and structural characterization of the introduced mutations are used to evaluate their success. Combination of individual stabilizing mutations proved to be beneficial, and a combinatorial construct was chosen for mouse immunizations in order to evaluate the immunogenicity of the antigen. The third chapter builds upon Chapter 2 through use of stabilized hMPV F to characterize the antibody repertoire from naturally infected elderly donors. Detailed mapping of hMPV F antigenic sites is achieved through evaluating binding competition with previously characterized antibodies, and structural techniques further identified four antigenic sites that were unable to be placed by the competition binding assay. Through stabilization of prefusion hMPV F in Chapter 2 and antibody isolation in Chapter 3, this work helps move us toward a vaccine and therapeutics for the prevention and treatment of human metapneumovirus.