Chimeric recombinant human metapneumoviruses with the nucleoprotein or phosphoprotein open reading frame replaced by that of avian metapneumovirus exhibit improved growth in vitro and attenuation in vivo

Abstract

Chimeric versions of recombinant human metapneumovirus (HMPV) were generated by replacing the nucleoprotein (N) or phosphoprotein (P) open reading frame with its counterpart from the closely related avian metapneumovirus (AMPV) subgroup C. In Vero cells, AMPV replicated to an approximately 100-fold-higher titer than HMPV. Surprisingly, the N and P chimeric viruses replicated to a peak titer that was 11- and 25-fold higher, respectively, than that of parental HMPV. The basis for this effect is not known but was not due to obvious changes in the efficiency of gene expression. AMPV and the N and P chimeras were evaluated for replication, immunogenicity, and protective efficacy in hamsters. AMPV was attenuated compared to HMPV in this mammalian host on day 5 postinfection, but not on day 3, and only in the nasal turbinates. In contrast, the N and P chimeras were reduced approximately 100-fold in both the upper and lower respiratory tract on day 3 postinfection, although there was little difference by day 5. The N and P chimeras induced a high level of neutralizing serum antibodies and protective efficacy against HMPV; AMPV was only weakly immunogenic and protective against HMPV challenge, reflecting antigenic differences. In African green monkeys immunized intranasally and intratracheally, the mean peak titer of the P chimera was reduced 100- and 1,000-fold in the upper and lower respiratory tracts, whereas the N chimera was reduced only 10-fold in the lower respiratory tract. Both chimeras were comparable to wild-type HMPV in immunogenicity and protective efficacy. Thus, the P chimera is a promising live HMPV vaccine candidate that paradoxically combines improved growth in vitro with attenuation in vivo.

FIG. 1.

Structures of the engineered N (A), P (B), and M (C) genes and flanking sequences and strategy for constructing HMPV/AMPV chimeras (D). The N and P genes and flanking sequences in panels A and B are shown with the HMPV construct on top and the HMPV/AMPV chimeric construct underneath. The BbsI, BsmBI, and BfuAI sites in panels A to C were introduced by PCR adapters to facilitate assembly; the MluI site (panel A) occurred naturally and the NheI site (panel C) had been introduced previously. Restriction sites are in italic type and underlined; the 4-nt compatible cohesive ends generated by cleavage with the respective enzymes are shown in bold italic type and underlined. The sequences of gene start (GS) and gene end (GE) signals are boxed. Panel D illustrates assembly of the N, P, and M fragments and shows the genomes of the resulting recombinants. HMPV genes are shown in white, and AMPV genes are shown in black. The synthetic SH gene (SHs) isgray.

FIG. 2.

Comparison of the multistep growth kinetics of rHMPV, rHMPV-N, rHMPV-P_A, and AMPV. Vero cells were infected at a MOI of 0.01 PFU per cell. At 24-h intervals, medium samples (0.5 ml of the 2-ml overlay) were taken and replaced by an equivalent volume of fresh medium containing 5 μg/ml of trypsin; in this case, the AMPV cultures also contained trypsin. The samples were analyzed by plaque assay on Vero cells. Each time point was represented by two wells, and each virus titration was done in duplicate. Means are shown. The detection limit was 5 PFU per ml.

FIG. 3.

Western blot analysis of viral proteins expressed in Vero cells infected at an input MOI of 1 PFU per cell with rHMPV (lane 1), rHMPV-N_A (lane 2), rHMPV-P_A (lane 3), or AMPV (lane 4) or mock infected (lane 5). The cells were harvested 72 h after infection, and lysates were prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and denaturing conditions using a 10% gel (NuPAGE; Invitrogen). After Western blot transfer to nitrocellulose, the membrane was incubated with a rabbit hyperimmune serum against purified HMPV followed by goat anti-rabbit antibodies conjugated with horseradish peroxidase, and the bound antibodies were visualized by chemiluminescence. The positions of the N, P, M, and M2-1 proteins are shown to the left of the gel. The positions of molecular mass markers (in kDa) are indicated to the right of the gel.

FIG. 4.

Kinetics of replication of rHMPV and AMPV chimera mutants in the upper and lower respiratory tracts of AGMs. Four animals per group (except for the mock-infected control group, which was composed of two animals) were inoculated by combined intranasal and intratracheal routes by using a 1-ml inoculum per site containing 10^6.0 PFU of the indicated virus on day 0. The nasopharyngeal swab (A) and tracheal lavage (B) specimens were taken on the indicated days, and the titer of virus shed was quantified by plaque assay. The detection limit was 0.7 log₁₀ PFU/ml.