Analysis of respiratory syncytial virus preclinical and clinical variants resistant to neutralization by monoclonal antibodies palivizumab and/or motavizumab

Abstract

Background: Palivizumab is a US Food and Drug Administration-approved monoclonal antibody for the prevention of respiratory syncytial virus (RSV) lower respiratory disease in high-risk infants. Motavizumab, derived from palivizumab with enhanced antiviral activity, has recently been tested in humans. Although palivizumab escape mutants have been generated in the laboratory, the development of resistant RSV in patients receiving palivizumab has not been reported previously.

Methods: We generated palivizumab and motavizumab escape mutants in vitro and examined the development of resistant mutants in RSV-breakthrough patients receiving immunoprophylaxis. The effect of these mutations on neutralization by palivizumab and motavizumab and in vitro fitness was studied.

Results: Antibody-resistant RSV variants selected in vitro had mutations at position 272 of the fusion protein, from lysine to asparagine, methionine, threonine, glutamine, or glutamate. Variants containing mutations at positions 272 and 275 were detected in breakthrough patients. All these variants were resistant to palivizumab, but only the glutamate variant at position 272 demonstrated resistance to motavizumab. Mixtures of wild-type and variant RSV soon lost the resistant phenotype in the absence of selection.

Conclusions: Resistant RSV variants were detected in a small subset (∼ 5%) of RSV breakthrough cases. The fitness of these variants was impaired, compared to wild-type RSV.

Figure 1.

Susceptibility of mutant variants to neutralization by palivizumab (A) and motavizumab (B). Microneutralization assays were performed in HEp-2 cells infected with respiratory syncytial virus (RSV) in the presence of serially diluted antibodies. Viral replication was measured using an F-protein–specific enzyme-linked immunosorbent assay (ELISA) represented by the A₄₅₀ value. The neutralization titer is presented as 50% inhibitory concentration (IC₅₀), the antibody concentration that gives a 50% reduction in the A₄₅₀ value, compared with the control wells without antibody.

Figure 2.

Growth kinetics of wild-type respiratory syncytial virus (RSV) A and mutant viruses. HEp-2 cells were inoculated at a multiplicity of infection of .1 pfu/cell with parental strain or mutant strains. The culture supernatant was sampled twice daily and titered for the presence of virus on HEp-2 cells using a 50% tissue culture infective dose (TCID₅₀) assay. A, Growth kinetics of parental RSV A Long and mutant variants A Long K272E, A Long K272Q, A Long K272M, A Long K272N, and A Long K272T. B, Growth kinetics of parental recombinant RSVA2 (rA2) and mutant viruses rA2 K272E, rA2 K272Q, rA2 S275F, and rA2 S275L. Growth capacity is represented as a log value of TCID₅₀ units per mL.

Figure 3.

Differential plaque neutralization focus forming assay determination of proportions of mutant variants during competitive replication with the A Long strain (A) or wild-type rA2 virus (B). Equal amounts of the parental respiratory syncytial virus (RSV) A and mutant variants were mixed and used to infect HEp-2 cells. A portion of virus-containing culture supernatant was passaged to new cells every 3.5 days. Remaining supernatants from each passage were stored at −80°C and tested by plaque neutralization focus forming assay in the absence or presence of palivizumab or motavizumab on Vero cells. The viral titer from the palivizumab containing plate represented palivizumab resistant virus that harbor the mutations K272Q, K272N, K272M, and K272T at antigenic site A of F protein. The viral titer from motavizumab containing plate represented mutant variant K272E that is resistant to motavizumab. The viral titer from the plain plate indicated both mutant and wild type virus. The percentage of mutant (resistant) virus of the total virus population (y-axis) was calculated by dividing the viral titer in the monoclonal antibody plate by that in the plate lacking monoclonal antibody.