Notably, protection appeared to be mediated by blocking the ability of PA to bind to its receptor since PEGylated antibody fragments exhibiting a KD=35 pM but lacking an Fc domain, and hence incapable of engaging innate immunity mechanisms of pathogen clearance, were protective 12. scAb (reference) and 4 replicates of 26.10 scAb (negative control). (e & f) Analysis of the four clones with the lowest dissociation rate constants (koff) as compared to 2C12.4. NIHMS509074-supplement-supplement_1.pdf (116K) GUID:?39719176-B863-4B69-957B-30C451889568 Abstract Genetic transfer of neutralizing antibodies has been shown to confer strong and persistent protection against bacterial and viral infectious agents. While it is well established that for many exogenous neutralizing antibodies increased antigen affinity correlates with protection, the effect of antigen affinity on antibodies produced following adenoviral gene transfer has not been examined. The mouse IgG2b monoclonal antibody 2C12.4 recognizes the Type III secretion apparatus protein LcrV (V antigen) and confers protection in mice when administered as an IgG intraperitoneally or, following genetic immunization with engineered, replication-defective serotype 5 human adenovirus (Ad) 1. 2C12.4 was expressed as a scFv fragment in and was shown to display a KD=3.5 nM by surface plasmon resonance (SPR) Dodecanoylcarnitine analysis. The 2C12.4 scFv was subjected to random mutagenesis and variants with increased affinity were isolated by flow cytometry using the Anchored Periplasmic Expression (APEx) bacterial display system. After a single round of mutagenesis, variants displaying up to 35-fold lower KD values (H8, KD=100 pM) were isolated. The variable domains of the H8 scFv were used to replace those of the parental 2C12.4 IgG encoded in the Ad vector, AdV giving rise to AdV.H8. The two adenoviral vectors resulted in similar titers of anti-V antigen antibodies 3 days post-immunization with 109, 1010 or 1011 particle units. Following intranasal challenge with 363 LD50CO92, 54% of the mice immunized with 1010 pu of AdV.H8 survived at the 14 day end point compared to only 15% survivors for the group Dodecanoylcarnitine immunized with AdV expressing Dodecanoylcarnitine the lower affinity 2C12.4 (P<0.04, AdV versus AdV.H8). These results indicate that affinity maturation of a neutralizing antibody delivered by genetic transfer may confer increased protection not only for challenge but possibly for other pathogens. Keywords: antibody, affinity maturation, is the etiologic agent of the plague and has been responsible for pandemic outbreaks occurring throughout the course of history. Although advances in our current living conditions, public health practices, and antibiotic therapies make future pandemics unlikely, outbreaks of plague resulting from biological warfare are a real threat. The features of that make it an attractive option for use as a biological weapon include availability of the organism, capacity for aerosol dissemination, potential for spread of secondary cases, and the high fatality rate of the pneumonic form of plague. In endemic regions of the world, the bacterium survives by causing chronic disease in animal reservoirs. It is spread among these animals and occasionally to humans predominantly through a flea vector, such as renders antibiotic therapy unreliable. For these reasons, is a likely agent to be used as a biological weapon since aerosolized bacteria can confer widespread pneumonic plague 2. Of the 11 species, only are human pathogens. is a gram-negative, non-motile, non-spore-forming bacterium that replicates intracellularly during the early stages of infection and grows predominantly extracellularly at later stages of the infectious cycle 2. At present, no plague vaccine has been approved for use in the US. Passive immunization with antibodies specific for the LcrV protein (V antigen) is an attractive alternative Dodecanoylcarnitine to vaccines and have been shown to be effective against lethal challenges with in a dose-dependent manner 1. For several neutralizing antibodies the degree of protection against challenge with Dodecanoylcarnitine pathogen correlates with antigen binding affinity 8-11. For example, while monoclonal antibodies and antibody fragments to the Protective Antigen (PA) of with a KD=11 nM fail to confer protection against challenge with the holotoxin or with intranasally administered spores, engineered antibody variants displaying 40- to 200-fold higher affinities Rabbit polyclonal to ALX3 were protective in different animal models 8,12. Notably, protection appeared to be mediated by blocking the ability of PA to bind to its receptor since PEGylated antibody fragments exhibiting a KD=35.
