Infection of nonhuman primates with recombinant human metapneumovirus lacking the SH, G, or M2-2 protein categorizes each as a nonessential accessory protein and identifies vaccine candidates

Abstract

Recombinant human metapneumovirus (HMPV) in which the SH, G, or M2 gene or open reading frame was deleted by reverse genetics was evaluated for replication and vaccine efficacy following topical administration to the respiratory tract of African green monkeys, a permissive primate host. Replication of the deltaSH virus was only marginally less efficient than that of wild-type HMPV, whereas the deltaG and deltaM2-2 viruses were reduced sixfold and 160-fold in the upper respiratory tract and 3,200-fold and 4,000-fold in the lower respiratory tract, respectively. Even with the highly attenuated mutants, there was unequivocal HMPV replication at each anatomical site in each animal. Thus, none of these three proteins is essential for HMPV replication in a primate host, although G and M2-2 increased the efficiency of replication. Each gene-deletion virus was highly immunogenic and protective against wild-type HMPV challenge. The deltaG and deltaM2-2 viruses are promising vaccine candidates that are based on independent mechanisms of attenuation and are appropriate for clinical evaluation.

FIG. 1.

Structures of the genomes of rHMPV and the ΔSH, ΔG, and ΔM2-2 gene deletion mutants. The wild-type rHMPV genome is shown at the bottom, drawn approximately to scale. The expanded view (not to scale) shows the M2-SH-G genome region. The genes are shown as open rectangles flanked on the upstream and downstream ends by the gene start (filled triangles) and gene end (filled boxes) transcription signals, respectively. The nucleotide length of each gene is listed underneath, and the calculated amino acid length of its encoded protein is given in italics above. Intergenic regions are shown as horizontal lines. The deletions in the ΔSH and ΔG viruses are indicated by dotted lines and involved 640 and 860 nucleotides, respectively (5). The M2-2 ORF was silenced by deleting 152 nucleotides and introducing stop codons (star) in the remnant ORF (8). Also shown are the NheI, BsiWI, and BsrGI sites together with their nucleotide positions in the antigenomic RNA sequence. The genome lengths of the recombinant viruses are indicated to the left.

FIG. 2.

Kinetics of replication of biologically derived HMPV CAN97-83, rHMPV, and the gene deletion mutants in the upper and lower respiratory tracts of African green monkeys. Four animals per group, except for the CAN97-83 and control (mock) groups composed of two animals, were inoculated by the combined intranasal and intratracheal routes by using a 1-ml inoculum per site containing 10^6.0 TCID₅₀ of the indicated virus on day 0. The nasopharyngeal swab (A) and tracheal lavage (B) specimens were taken on the indicated days, and the titers of shed virus were quantified by plaque assay. The detection limit was 0.7 log₁₀ PFU/ml.