Direct Sequencing of Antibodies of the Influenza Immune Response

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Abstract The annual epidemics of influenza result in a substantial number of hospitalizations with an estimated 3 to 5 million cases of severe disease, and 300,000 to 500,000 deaths globally. During the 20th century, three major influenza pandemics have occurred with a total mortality of 50–100 million people. Recently, new influenza monoclonal antibodies (mAbs) have been identified that are capable of neutralizing a wide range of viruses. In the last eight years, several broadly neutralizing, stem-reactive antibodies have been identified. Of these stem antibodies, only one recognized all influenza A subtypes however this breadth of recognition was only elicited after in vitro mutagenesis. This suggests that an epitope to guide a universal vaccine strategy has yet be defined. Stem antibodies could provide universal influenza coverage since they are potentially capable of neutralizing both influenza A and B viruses. One strategy to identify neutralizing stem-specific antibodies involves immortalized B cells from immunized individuals. B cell-derived mAbs that cross react with the H5 and H7 HA subtypes have been shown to yield high affinity stem-specific mAbs that can bind to all influenza A HA subtypes. An alternative strategy, that doesn’t involve B cell cloning, starts with an antigen specific population of stem binding polyclonal antibodies (pAbs) that can then be directly sequenced by proteomic tandem mass spectrometry (MS/MS). These pAbs are a preferred source for mAb discovery since the peripheral antibody population, not the peripheral B cell population, is representative of the full humoral immune response. Direct sequencing of peripheral pAbs can rapidly mine this immune diversity to yield mAbs against novel and unique epitopes.