Rational Design of Human Metapneumovirus Live Attenuated Vaccine Candidates by Inhibiting Viral mRNA Cap Methyltransferase

Abstract

The paramyxoviruses human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), and human parainfluenza virus type 3 (hPIV3) are responsible for the majority of pediatric respiratory diseases and inflict significant economic loss, health care costs, and emotional burdens. Despite major efforts, there are no vaccines available for these viruses. The conserved region VI (CR VI) of the large (L) polymerase proteins of paramyxoviruses catalyzes methyltransferase (MTase) activities that typically methylate viral mRNAs at positions guanine N-7 (G-N-7) and ribose 2=-O. In this study, we generated a panel of recombinant hMPVs carrying mutations in the S-adenosylmethionine (SAM) binding site in CR VI of L protein. These recombinant viruses were specifically defective in ribose 2=-O methylation but not G-N-7 methylation and were genetically stable and highly attenuated in cell culture and viral replication in the upper and lower respiratory tracts of cotton rats. Importantly, vaccination of cotton rats with these recombinant hMPVs (rhMPVs) with defective MTases triggered a high level of neutralizing antibody, and the rats were completely protected from challenge with wild-type rhMPV. Collectively, our results indicate that (i) amino acid residues in the SAM binding site in the hMPV L protein are essential for 2=-O methylation and (ii) inhibition of mRNA cap MTase can serve as a novel target to rationally design live attenuated vaccines for hMPV and perhaps other paramyxoviruses, such as hRSV and hPIV3. IMPORTANCEHuman paramyxoviruses, including hRSV, hMPV, and hPIV3, cause the majority of acute upper and lower respiratory tract infections in humans, particularly in infants, children, the elderly, and immunocompromised individuals. Currently, there is no licensed vaccine available. A formalin-inactivated vaccine is not suitable for these viruses because it causes enhanced lung damage upon reinfection with the same virus. A live attenuated vaccine is the most promising vaccine strategy for human paramyxoviruses. However, it remains a challenge to identify an attenuated virus strain that has an optimal balance between attenuation and immunogenicity. Using reverse genetics, we generated a panel of recombinant hMPVs that were specifically defective in ribose 2=-O methyltransferase (MTase) but not G-N-7 MTase. These MTase-defective hMPVs were genetically stable and sufficiently attenuated but retained high immunogenicity. This work highlights a critical role of 2=-O MTase in paramyxovirus replication and pathogenesis and a new avenue for the development of safe and efficacious live attenuated vaccines for hMPV and other human paramyxoviruses.

Zinc Binding Activity of Human Metapneumovirus M2-1 Protein Is Indispensable for Viral Replication and Pathogenesis In Vivo

“…However, these tissues were positive for hMPV RNA (data not shown), as determined by realtime RT-PCR, which is not surprising, because real-time RT-PCR was much more sensitive than plaque assay. Similar results were observed for other hMPV mutants, such as receptor binding-defective rhMPVs and methyltransferase-defective rhMPVs, which were defective in receptor binding activity in fusion (F) protein and mRNA cap methyltransferase activity in the large (L) polymerase protein, respectively (31,35). Another possibility is that infectious hMPV has been cleared from nasal turbinate and lungs for these mutants at day 4 postinfection.…”

“…Plasmids encoding the hMPV minigenome, the full-length genomic cDNA of hMPV strain NL/ 1/00, and support plasmids expressing the hMPV N protein (pCITE-N), P protein (pCITE-P), L protein (pCITE-L), and M2-1 protein (pCITE-M2-1) were kindly provided by Ron A. M. Fouchier at the Department of Virology, Erasmus Medical Center, Rotterdam, the Netherlands. The F cleavage site in the genome of hMPV NL/1/00 was modified to a trypsinindependent F cleavage site, as described previously (31,32). The M2-1 mutants of hMPV were generated by site-directed mutagenesis using the QuikChange methodology (Stratagene, La Jolla, CA).…”

“…rhMPVs were rescued using a reverse genetics system as described previously (27,31). Briefly, BHK-SR19-T7 cells (kindly provided by Apath LLC), which stably express T7 RNA polymerase, were transfected with 5.0 g of plasmid phMPV carrying the full-length hMPV genome, 2.0 g of pCITE-N, 2.0 g of pCITE-P, 1.0 g of pCITE-L, and 1.0 g of pCITE-M2-1 using Lipofectamine 2000 (Life Technologies).…”

Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis

“…hMPV-specific neutralizing antibody titers were determined using a plaque reduction neutralization assay (21,28). Briefly, cotton rat sera were collected by retro-orbital bleeding weekly until challenge.…”

“…Similar to hRSV, inactivated vaccines are not suitable for hMPV because it causes enhanced lung damage upon reinfection by the same virus in animal models (47). In contrast, enhanced lung damage has not been observed for live attenuated vaccine candidates in animal models (28,(48)(49)(50). Thus, significant efforts have been devoted to developing live attenuated vaccines for hMPV.…”