Analysis of Respiratory Syncytial Virus Preclinical and Clinical Variants Resistant to Neutralization by Monoclonal Antibodies Palivizumab and/or Motavizumab

Respiratory syncytial virus (RSV) is a member of the Pneumovirus genus in the Paramyxoviridae family. The RSV genome consists of a negative-sense, nonsegmented, single strand of RNA encoding 10 proteins [1]. RSV is an enveloped virus, and its antigenicity is determined by 2 transmembrane glycoproteins, the attachment glycoprotein (G) and the fusion (F) protein. RSV is classified into A and B subgroups, originally based on antigenic differences in the G protein [2]. RSV is the most serious respiratory pathogen in infants and young children, causing annual epidemics of bronchiolitis and pneumonia worldwide [1, 3, 4]. Overall, RSV infection is responsible for ∼2% of the hospitalization rate among infants <1 year of age [5]. This rate increases at least 4–5-fold among children at high risk of severe RSV disease, including premature infants and those with chronic lung disease of prematurity, immunodeficiency, or complicated congenital heart disease [6–12]. RSV infection in infants and children can cause lung function deterioration that may be sustained for months after the acute illness, and in some circumstances, years of recurrent wheezing or asthma may ensue [13, 14]. To date, prevention of RSV disease has only been achieved by the passive administration of RSV-specific immunoglobulin. Prophylaxis with palivizumab (MedImmune), a humanized monoclonal antibody (mAb) that is directed against the RSV F protein, can significantly reduce the rate of RSV-related hospitalizations in high-risk infants [15, 16]. Motavizumab (MEDI-524; MedImmune), an enhanced mAb developed by affinity maturation of palivizumab [17–19], is in clinical development. Nonclinical studies demonstrated that motavizumab was more effective than palivizumab at neutralizing RSV in vitro. In addition, at equivalent serum and lung levels, motavizumab was shown to be superior to palivizumab at reducing RSV infection in both the upper and lower airways of cotton rats [18]. Motavizumab and palivizumab bind antigenic site A, a highly conserved region on the RSV F protein between amino acids 258 and 275 [20]. Similar to other RNA viruses, replication of RSV depends on an RNA polymerase that lacks proofreading and repair capability, resulting in a relatively high mutation rate. This mutability could increase the potential for the generation of resistant mutants under selective drug pressure, such as antibody prophylaxis. In vitro development of RSV A mutants resistant to palivizumab has been reported previously. Beeler and Coelingh [20] isolated RSV monoclonal antibody resistant mutants (MARMs) containing phenotypic amino acid variations at positions 262, 275, and 276 of the F protein with use of the murine precursor to palivizumab, mAb1129. Sullender et al [21–23] also isolated palivizumab MARMs containing mutations at positions 268 and 272. The potential for resistance to occur was also explored during the clinical development of palivizumab. In a prospective study using a binding assay that was predictive of palivizumab neutralization, the investigators showed that palivizumab bound to all 25 RSV isolates collected from patients actively receiving palivizumab [24]. However, the number of samples was small in this study, and the assay was suboptimal for detecting minor drug-resistant viral populations. Recently, nucleotide sequence analysis of RSV isolates collected directly from nasal wash specimens from infants who received palivizumab and still developed acute lower tract respiratory infection revealed an F protein mutation at position 272 from lysine (K) to glutamate (E). Although the susceptibility of this variant to neutralization by palivizumab could not be determined because it did not propagate in cell culture [25], it was suggested that this K272E variant would most likely be less susceptible to neutralization by palivizumab, because multiple in vitro-selected palivizumab MARMs contain mutations at this position. To date, the data available on the rate of emergence of clinical resistant variants during treatment are limited. In the present study, we describe the in vitro selection and characterization of additional palivizumab MARMs and a novel motavizumab MARM. We also examined amino acid changes in the F protein of RSV isolates collected from RSV-breakthrough patients receiving palivizumab or motavizumab in a large phase 3 clinical study comparing motavizumab with palivizumab (MI-CP110: Study of MEDI-524; for the prophylaxis of RSV disease in high risk children) [26]. The sensitivity of these immunoprophylaxis breakthrough isolates to motavizumab and palivizumab was determined using recombinant RSV (rRSV). Moreover, the effect of these neutralization-resistant mutations on in vitro fitness was assessed.