In vitro and in vivo fitness of respiratory syncytial virus monoclonal antibody escape mutants

Abstract

Respiratory syncytial virus (RSV) is the only infectious disease for which a monoclonal antibody (MAb) is used in humans. Palivizumab (PZ) is a humanized murine MAb to the F protein of RSV. PZ-resistant viruses appear after in vitro and in vivo growth of RSV in the presence of PZ. Fitness for replication could be a determinant of the likelihood of dissemination of resistant viruses. We assessed the fitness of two PZ-resistant viruses (F212 and MP4). F212 grew less well in cell culture than the parent A2 virus and was predicted to be less fit than A2. Equal amounts of F212 and A2 were mixed and passaged in cell culture. F212 disappeared from the viral population, indicating it was less fit than the A2 virus. The MP4 virus grew as well as A2 in culture and in cotton rats. A2/MP4 virus input ratios of 1:1, 10:1, 100:1, and 1,000:1 were compared in competitive replication. For all input ratios except 1,000:1, the MP4 virus became dominant, supplanting the A2 virus. The MP4 virus also dominated the A2 virus during growth in cotton rats. Thus, the mutant MP4 virus was more fit than A2 virus in both in vitro and in vivo competitive replication. Whether this fitness difference was due to the identified nucleotide substitutions in the F gene or to mutations elsewhere in the genome is unknown. Understanding the mechanisms by which mutant virus fitness increased or decreased could prove useful for consideration in attenuated vaccine design efforts.

FIG. 1.

Differential plaque assay determination of mutant virus proportions during competitive replication with parent A2 virus. (A) Equal amounts of the parent A2 and the mutant F212 or (B) different initial A2/MP4 ratios (1:1, 10:1, 100:1, and 1,000:1) of the parent A2 and the mutant MP4 were mixed and used to infect HEp-2 cells. Infected-cell medium was passaged to new cells and at the passage numbers shown (x axis) tested by differential plaque assay to determine the proportions of mutant virus present. Duplicate plaque assays were performed on Vero cells, with and without PZ (4 μg/ml) in the agar overlay. The viral titer from the PZ overlay plate represented mutant virus that was resistant to PZ; the viral titer from the plain overlay plate represented both mutant and wild-type A2 virus. The proportion of mutant virus in the mixed population (y axis) was obtained by dividing the viral titer in the PZ plate by that in the no-PZ plate.

FIG. 2.

Restriction fragment analysis of the dynamics of mutant F212 virus during competitive replication with parent A2 virus. Shown are RT-PCR-amplified DNAs from sequentially passaged A2/F212 mixed viruses (passage numbers shown as p1, p2, etc.) and control A2 and F212 viruses incubated with MfeI and separated by agarose gel electrophoresis. The first lane (standard [std]) is a 100-bp DNA ladder with 200-bp and 600-bp sizes indicated. The A2 virus did not cleave (lane A2), whereas the mutation in the F212 F gene introduced an MfeI site, resulting in two bands after digestion (lane F212). The presence of three bands indicates a mixture of A2 and F212, and a single upper band indicates the presence of A2 without detection of F212.

FIG. 3.

Dynamics of mutant F212 virus during competitive replication with parent A2 virus assessed by nucleotide sequence analysis. Competitive replication was performed beginning with (A) a 1:1 ratio of the A2 and F212 viruses, or (B) different initial ratios of A2 to MP4 (1:1, 10:1, 100:1, and 1000:1) of the parent A2 and the mutant MP4 were mixed and used to infect HEp-2 cells and passaged sequentially. At the passage numbers shown (x axis), cell lysates were taken for RT-PCR and nucleotide sequence determination. The relative amounts of (A) F gene nucleotide 816 (A from A2 virus and T from mutant F212) or (B) F gene nucleotide position 828 (A from A2 virus and T from mutant MP4) were estimated using Edit View (Applied Biosystems, Inc.), and the proportion of mutant virus in the mixture (T/A + T) is shown (y axis).

FIG. 4.

Dynamics of mutant MP4 virus during competitive replication with parent A2 virus with input mixtures of different multiplicities of infection. Equal amounts of mutant MP4 and A2 virus (by PFU) were mixed and used to infect HEp-2 cells with different initial MOIs (0.1, 0.5, 1, and 2), and infected-cell medium was passaged sequentially to new cells. At the passage numbers shown (x axis), the relative proportion (y axis) of the MP4 virus was determined by differential plaque assay as described in the legend to Fig. 2.