High mutant frequency in populations of a DNA virus allows evasion from antibody therapy in an immunodeficient host

Abstract

The degree of genetic heterogeneity of DNA virus populations in nature and its consequences for disease control are virtually unknown. The parvovirus minute virus of mice (MVMi) was used here to investigate (i) the frequency of antibody-escape mutants in populations of a DNA virus and (ii) the ability of a DNA virus to evade in the long-term a passive monoclonal antibody (MAb) therapy in an immunodeficient natural host. Independent clonal populations of MVMi harbored a high proportion of mutants resistant to neutralizing MAb (mutant frequency = [2.8 +/- 0.5] x 10(-5)) that rapidly evolved under antibody pressure in culture to become mixtures dominated by genotypically diverse escape mutants. Immunodeficient mice naturally infected with clonal populations of MVMi and subsequently treated by intravenous injections of MAb were initially protected from the characteristic viral induced lethal leukopenia. However, some treated animals developed a delayed severe leukopenic syndrome associated with the emergence of genetically heterogeneous populations of MAb-resistant mutants in the MVMi main target organs. The 11 plaque-purified viruses analyzed from an antibody-resistant population obtained from one animal corresponded to four different mutant genotypes, although their consensus sequence remained wild type. All cloned escape mutants harbored single radical amino acid changes within a stretch of seven residues in a surface-exposed loop at the threefold axes of the capsid. This antigenic site, which can tolerate radical changes preserving MVMi pathogenic potential, may thereby allow the virus to evade the immune control. These findings indicate a high genetic heterogeneity and rapid adaptation of populations of a mammal DNA virus in vivo and provide a genetic basis for the failure of passive immunotherapy in the natural host.

FIG. 1.

Proportion of MAR mutants in populations of MVMi. Independent clonal populations of MVMi (stocks A and B) obtained in the absence of antibody as described in Materials and Methods were used to infect 10⁶ NB324K cells at a multiplicity of infection of 1. At 4 h postadsorption, MAb B7 was added to the medium (2 Nu/ml), and the intracellular virus was harvested at 72 hpi (passage 1). Passages 2 and 3 were similarly obtained in the presence of antibody by inoculating 10⁶ cells with one-tenth of the virus obtained in the previous passage. For each population, the total infectious virus titer (bars) and the MAR mutants titer (white portion of each bar) were determined by plaque assay in the absence or presence of antibody, respectively. The phenomenon was also observed with MVM-neutralizing MAb harvested from other hybridoma cell lines (not shown).

FIG. 2.

Protection analysis of a passive immunotherapy in SCID mice lethally infected by MVMi. Animals 1 to 10 and negative controls (n = 8) were oronasally infected with 10⁶ PFU of MVMi and treated at the indicated dates (arrows) with an intravenous injection of 12 Nu of MAb B7. Bars represent the life span of the animals, and the shaded portions indicate the appearance of the leukopenic syndrome (white blood cell count, <5 × 10⁵/ml of peripheral blood).

FIG. 3.

Phenotype of viral populations recovered from MVMi-infected SCID mice. (A) Neutralization kinetics comparing a MVMi stock with virus populations obtained from the spleen of untreated mice (control) and of some MAb-treated mice (animals 4, 8, 9, and 10). Shown are the percent residual infectivity values after incubation with or without the indicated units of MAb B7. The experiment was repeated three times with similar outcomes. (B) Representative plaque assay showing MAR mutants recovered from spleen homogenates of the indicated mice. Note the different proportion of MAR mutants between samples.

FIG. 4.

Sequence analysis of mutations conferring MAb B7 resistance in vivo or in vitro. (A) Nucleotide sequence of MVMi populations recovered from the spleen of the indicated mice. Shown is the VP region of MVM genome where VP coding changes map. The predominant point mutation in the population of mouse 4 and mouse 10 is indicated (arrows). A punctual change V551A found in some mouse organs but not correlating with MAb resistance is not indicated in the figure. (B) Scheme of the VP1 and VP2 structural proteins translated from alternative spliced messengers expressed from the P38 viral promoter. The sequence of the segment of both polypeptides where MAR mutations were located is shown below by using the VP2 numbering. Amino acid differences relative to the parental sequence in viral clones isolated from the indicated mice or from populations independently obtained in vitro (experiments 1 and 2 [see Fig. 1, Exp. 1 and Exp. 2]) are indicated by using the single-letter code. The letter n refers to the number of clones from each population showing the corresponding amino acid substitution.

FIG. 5.

Localization in the MVMi capsid structure of the amino acid residues whose replacement conferred the MAR phenotype. (A) Model of the complete capsid. (B) Close-up of one of the protruding spikes located at the threefold axes. The amino acid residues that form the spike are represented as space-filling models and colored white, except for those involved in antibody recognition (Fig. 4), which are colored red. All other capsid residues are colored blue and are shown either as wireframe (A) or ribbon (B) models. The figure was prepared by using the program RasMol (53) and the MVMi coordinates (1MVM) deposited in the PDB (1).