To determine the degree of cross-reactivity at the level of individual antibodies, we analyzed binding of our clones to various influenza HA antigens (Fig

To determine the degree of cross-reactivity at the level of individual antibodies, we analyzed binding of our clones to various influenza HA antigens (Fig. detailed immunochemical analysis of individual human solutions to virus neutralization in the setting of an actual virulent influenza outbreak. Remarkably, three of these antibodies neutralized both H1 RO-9187 and H5 subtype influenza viruses. Newly emergent, highly pathogenic influenza virus strains pose a profound threat to man. Three influenza pandemics have occurred within the past 100 years, each with devastating consequences (1). The recent emergence of the H5N1 virus sub-type, although mainly confined at present to avian hosts, has already demonstrated virulence in humans, causing the death of >200 people (2). Therefore, health care officials, researchers, and governments are actively considering their options should a pandemic occur. One widely considered approach concerns the use of passive immunization either for the prevention of disease or for treatment after exposure to virus (3). The potential for passive immunization against influenza has been evident since the Spanish influenza outbreak nearly a century ago, where the benefits of RO-9187 transfused blood, sera, and blood products reduced the risk of mortality by >50% (3). Recently, the benefits of treatment with convalescent plasma in instances of H5N1 influenza have also been reported (4, 5). Additionally, passive immunization with human and mouse monoclonal antibodies has been reported to protect animals from death, even when administered after H5N1 infection (6). The most logical source of human antibodies for passive therapy would be patients who have survived infection. With modern combinatorial antibody library technologies, it is now possible to capture the entire immunological history of an individual’s response to an infection (7, 8). Because antibody libraries contain the RO-9187 complete record of an individual’s response to pathogens, one can recover the repertoire specific to a given agent by using a laboratory process of selective enrichment. Such libraries give archival information about the nature of antibodies made during the infection and allow recovery of potentially therapeutic human monoclonal antibodies. Importantly, antibody recovery is independent of whether an active antibody response is still occurring at the time the sample is taken. Thus, depending on when the libraries are constructed, one may obtain antibodies that are currently being made and/or are part of the individual’s immunological history. For infections that may be lethal, such analyses carried out on surviving patients may be particularly important because they chart some of the immunological mechanisms used during a successful host defense in the actual clinical setting of an outbreak. Typically, when libraries are prepared from individuals who have been infected with a virus, hundreds to thousands of different antibodies Rabbit Polyclonal to Dipeptidyl-peptidase 1 (H chain, Cleaved-Arg394) are obtained, as opposed to only a few when other methods are used (8). A comparative sequence analysis of these antibodies allows a detailed map of the chemistry of antibody binding. Similarly, a comparison of neutralizing and nonneutralizing antibodies can give important information about the nature of binding interactions that are critical to neutralization. Here we describe the creation of comprehensive avian influenza antibody libraries made from survivors of infection with an avian influenza virus during a confirmed outbreak. We have used these libraries to obtain large numbers of monoclonal antibodies to the H5N1 avian influenza virus, some of which have broad reactivity and are neutralizing across viral subtypes. Ultimately, combinatorial antibody libraries may hold the key to immunotherapy, such as passive immunization using one or more member antibodies, or they may guide the development of vaccines directed at the antigenic target(s) of the neutralizing antibodies in the library. Results The Outbreak and Source of Material. Between December 2005 and January 2006, an outbreak of avian influenza H5N1 occurred in Turkey (9). In total, 12 individuals were infected and only 8 survived. Because bone marrow RNA contains the archived record of all antibodies made by an individual, we selected it as our source material. We obtained bone serum and marrow from six from the Turkish survivors after their recovery and successfully.