Affinity maturation of an anti-V antigen IgG expressed in situ through adenovirus gene delivery confers enhanced protection against Yersinia pestis challenge

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 in situ following adenoviral gene transfer has not been examined. The mouse IgG2b monoclonal antibody 2C12.4 recognizes the Yersinia pestis 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 E. coli and was shown to display a KD=3.5 nM by surface plasmon resonance (SPR) 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, AdαV giving rise to AdαV.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 LD50Y. pestis CO92, 54% of the mice immunized with 1010 pu of AdαV.H8 survived at the 14 day end point compared to only 15% survivors for the group immunized with AdαV expressing the lower affinity 2C12.4 (P<0.04, AdαV versus AdαV.H8). These results indicate that affinity maturation of a neutralizing antibody delivered by genetic transfer may confer increased protection not only for Y. pestis challenge but possibly for other pathogens.

Plague Vaccines: Status and Future

“…Both constructs elicited strong humoral immunity in mice immunized intramuscularly with greater efficacy than an injection of adjuvanted purified V or F1 [191]. In addition, they also employed adenovirus (Ad) to deliver monoclonal antibodies (MAbs) specific for the Y. pestis LcrV antigen, which provided good protection for immunized mice against intranasal challenge with 363 LD 50 of Y. pestis CO92 [192, 193]. …”

Monoclonal Antibodies Recognizing the Surface Autolysin IspC of Listeria monocytogenes Serotype 4b: Epitope Localization, Kinetic Characterization, and Cross-Reaction Studies

“…The affinities of the MAbs from this study, in the nanomolar range, are within the biologically useful range for current diagnostic tests such as ELISA and M2773, M2775, M2781, M2792 and M2797 have a particularly high affinity compared to other MAbs produced using a similar methodology [26], [27], [28]. In a clinical setting, high-affinity MAbs have a greater neutralizing potential than low-affinity MAbs during passive immunization [29]. Future work with these selected MAbs against L. monocytogenes serotype 4b will concentrate on the ability of these MAbs to diagnose this important serotype using novel, culture independent, diagnostic platforms currently under development by our group.…”

Antibodies Inhibiting the Type III Secretion System of Gram-Negative Pathogenic Bacteria

“…Abs targeting LcrV were sufficient to prevent translocation of effector proteins by the Y. pestis T3SS [ 84 ]. Since this time, there has been an explosion of research regarding anti-LcrV antibodies [ 72 , 85 , 86 , 87 , 88 , 89 , 90 ].…”