Discovery of a Prefusion Respiratory Syncytial Virus F-Specific Monoclonal Antibody That Provides Greater In Vivo Protection than the Murine Precursor of Palivizumab

Abstract

Palivizumab, a humanized murine monoclonal antibody that recognizes antigenic site II on both the prefusion (pre-F) and postfusion (post-F) conformations of the respiratory syncytial virus (RSV) F glycoprotein, is the only prophylactic agent approved for use for the treatment of RSV infection. However, its relatively low neutralizing potency and high cost have limited its use to a restricted population of infants at high risk of severe disease. Previously, we isolated a high-potency neutralizing antibody, 5C4, that specifically recognizes antigenic site Ø at the apex of the pre-F protein trimer. We compared in vitro and in vivo the potency and protective efficacy of 5C4 and the murine precursor of palivizumab, antibody 1129. Both antibodies were synthesized on identical murine backbones as either an IgG1 or IgG2a subclass and evaluated for binding to multiple F protein conformations, in vitro inhibition of RSV infection and propagation, and protective efficacy in mice. Although 1129 and 5C4 had similar pre-F protein binding affinities, the 5C4 neutralizing activity was nearly 50-fold greater than that of 1129 in vitro. In BALB/c mice, 5C4 reduced the peak titers of RSV 1,000-fold more than 1129 did in both the upper and lower respiratory tracts. These data indicate that antibodies specific for antigenic site Ø are more efficacious at preventing RSV infection than antibodies specific for antigenic site II. Our data also suggest that site Ø-specific antibodies may be useful for the prevention or treatment of RSV infection and support the use of the pre-F protein as a vaccine antigen.IMPORTANCE There is no vaccine yet available to prevent RSV infection. The use of the licensed antibody palivizumab, which recognizes site II on both the pre-F and post-F proteins, is restricted to prophylaxis in neonates at high risk of severe RSV disease. Recommendations for using passive immunization in the general population or for therapy in immunocompromised persons with persistent infection is limited because of cost, determined from the high doses needed to compensate for its relatively low neutralizing potency. Prior efforts to improve the in vitro potency of site II-specific antibodies did not translate to significant in vivo dose sparing. We isolated a pre-F protein-specific, highpotency neutralizing antibody (5C4) that recognizes antigenic site Ø and compared its efficacy to that of the murine precursor of palivizumab (antibody 1129) matched for isotype and pre-F protein binding affinities. Our findings demonstrate that epitope specificity is an important determinant of antibody neutralizing potency, and defining the

Alternative conformations of a major antigenic site on RSV F

“…Previous studies have demonstrated that AM22 and RSD5 potently neutralize RSV and preferentially bind to the prefusion RSV F conformation, similar to the previously characterized site Ø antibodies D25 and 5C4 [8,35,38,42]. However, differences in antibody kinetics and subtype specificities have not been fully explored.…”

“…Upon triggering, site Ø undergoes an extensive structural rearrangement in which α4 and the α4-α5 loop refold to form the continuous α5-helix observed in the postfusion F conformation [8]. Comparison of the neutralization potency of two site Ø antibodies, D25 [34] and 5C4 [35], with palivizumab, a site II-directed conformation-independent antibody [36], demonstrated that the prefusionspecific antibodies are 10-100 times more potent [8]. Other potent prefusion-specific human antibodies that bind to the apex of the trimer, such as AM22 and RSD5, have also been isolated in recent years [8,37,38], and one of them (MEDI8897) is now in advanced stages of clinical development [39].…”

The Optimal Concentration of Formaldehyde is Key to Stabilizing the Pre-Fusion Conformation of Respiratory Syncytial Virus Fusion Protein

“…Lung homogenates were made using a gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) on program Lung 02. Pulmonary virus titration was determined via plaque assay, and replication of the RSV A2 genome RNA was detected by measuring the target RSV A2 N gene via qPCR on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) as previously described [15,16]. The left lungs were kept in 10% formaldehyde and paraffin-embedded.…”

Vaccine Update: Recent Progress With Novel Vaccines, and New Approaches to Safety Monitoring and Vaccine Shortage

“…Specific antigenic sites of interest include antigenic site 0 and site II, and multiple products are in early phases of development, from discovery to preclinical. 52,66 Nanobodies are described as small antibody fragments that consist of a single monomeric variable domain of the heavy chain, and which have antigen-binding capacity and similar activity as conventional antibodies. However, they are easier to produce and their small size and stability permits the creation of multivalent constructs to bind to specific antigenic sites and the option of mucosal (intranasal/inhaled) instead of systemic administration.…”