Viral Fitness Can Influence the Repertoire of Virus Variants Selected by Antibodies Verónica Martín, Celia Perales, Mercedes Dávila and Esteban Domingo⁎ Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Cantoblanco, E-28049 Madrid, Spain Minority genomes in the mutant spectra of viral quasispecies may differ in relative fitness. Here, we report experiments designed to evaluate the contribution of relative fitness to selection by a neutralizing monoclonal antibody (mAb). We have reconstructed a foot-and-mouth disease virus (FMDV) quasispecies, with two matched pairs of distinguishable mAb- escape mutants as minority genomes of the mutant spectrum. Each mutant of a pair differs from the other by 11-fold or 33-fold in relative fitness. Analysis of the mutant spectra of virus populations selected with different concentrations of antibody in infections in liquid culture medium has documented a dominance of the high fitness counterpart in the selected population. Plaque development as a function of increasing concentration of the antibody has shown that each mutant of a matched pair yielded the same number of plaques, although the high fitness mutant required less time for plaque formation, and attained a larger plaque size at any given time-point. This result documents equal intrinsic resistance to the antibody of each mutant of a matched pair, confirming previous biochemical, structural, and genetic studies, which indicated that the epitopes of each mutant pair were indistinguishable regarding reactivity with the mono- clonal antibody. Thus, relative viral fitness can influence in a significant way the repertoire of viral mutants selected from a viral quasispecies by a neutralizing antibody. We discuss the significance of these results in relation to antibody selection, and to other selective forces likely encountered by viral quasispecies in vivo. © 2006 Elsevier Ltd. All rights reserved. *Corresponding author Keywords: viral quasispecies; mutant spectrum; selection dynamics; competition; foot-and-mouth disease virus Introduction Quasispecies dynamics during RNA virus replica- tion involves the continuous generation of variant genomes, competition among them, and selection of the fittest mutant distributions in response to selective forces. 1–3 The adaptive, cellular and hu- moral immune response of the host organisms is a selective constraint operating during virus replica- tion in vivo. Viruses may modulate, disrupt or evade, through genetic variation, the host immune res- ponse. Escape from cytotoxic T cells or from neutra- lizing antibodies may contribute to viral persistence and disease progression. 4–11 Several mechanisms have been proposed for the neutralization of viral infectivity by antibodies. For naked viruses such as the picornaviruses, mechanisms can be divided into those that involve alteration, stabilization or aggre- gation of virions, and those that involve coating of the particle with antibody molecules to prevent attachment to cells. 12–15 Mutants that escape neutrali- zation by a monoclonal antibody (mAb) generally include an amino acid substitution at the relevant epitope, resulting in a decrease of affinity of the epi- tope for its corresponding paratope in the antibody molecule. 15 Several constraints may limit the types and location of the amino acid replacements that confer decreased sensitivity to an antibody. One constraint stems from codon usage and the amino acid re- placements compatible with a single point mutation within each of the codons that specify the residues relevant to the epitope–paratope interaction (two Abbreviations used: mAb, monoclonal antibody; FMDV, foot-and-mouth disease virus; pfu, plaque-forming units. E-mail address of the corresponding author: edomingo@cbm.uam.es doi:10.1016/j.jmb.2006.06.077 J. Mol. Biol. (2006) 362, 44–54 0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.