Abstract
Human metapneumovirus (HMPV) and human parainfluenza virus 3 (HPIV3) are among the leading causes of severe pediatric respiratory illness worldwide, and there are no licensed vaccines. Here, we describe live-attenuated pediatric intranasal vaccine candidates for dual protection against HMPV and HPIV3. An attenuated chimeric bovine/HPIV3 (B/HPIV3) based on bovine PIV3, with fusion (F) and hemagglutinin-neuraminidase surface glycoproteins replaced by those of HPIV3, was designed to express HMPV subgroup A F protein, the major protective HMPV antigen. Eight viruses were generated, each containing a different version of the HMPV F ORF, including wild-type (wt), codon-optimized (BBopt or GSopt) to increase protein expression, a chimeric form (GSopt-TMCT) with the transmembrane/cytoplasmic tail (TMCT) sequence replaced with that of BPIV3 F to increase HMPV F packaging, or versions with amino acid substitutions (BBopt-D185P, GSopt-D185P, BBopt-N46V/T160F, and GSopt-N46V/T160F) to improve stability in pre-fusion form and enhance immunogenicity. In hamsters, a single intranasal dose conferred serum HMPV- and HPIV3-neutralizing antibodies and provided protection in the upper and lower respiratory tract against HMPV challenge, similar to that afforded by wt HMPV infection. Control hamsters immunized intramuscularly with two doses of HMPV pre-fusion subunit antigens stabilized by D185P or N46V/T160F mutations developed HMPV-neutralizing antibody titers, similar to those induced by the B/HPIV3/HMPV-F candidates, and were similarly protected against HMPV challenge in the lungs. However, in the upper respiratory tract, B/HPIV3/HMPV-F candidates were significantly more protective than the subunit antigens. Thus, B/HPIV3 expressing HMPV wt or pre-fusion F are promising pediatric vaccine candidates for intranasal immunization against HMPV and HPIV3. IMPORTANCE: Human metapneumovirus (HMPV) and human parainfluenza virus 3 (HPIV3) are important pediatric pathogens and need effective vaccines. We developed live-attenuated chimeric bovine/HPIV3 (B/HPIV3) expressing the fusion (F) protein of HMPV subgroup A as a bivalent pediatric intranasal vaccine against HPIV3 and HMPV. To enhance HMPV F immunogenicity, three approaches were evaluated: codon optimization to increase expression, structure-based mutations to stabilize HMPV F in pre-fusion conformation, or replacement of the HMPV F transmembrane/cytoplasmic domains with those of BPIV3 F to increase incorporation in the virus particle. In hamsters, a single intranasal dose of any of the B/HPIV3s expressing HMPV F was as immunogenic and protective against HMPV as a prior HMPV infection. In the upper respiratory tract, these provided greater protection against HMPV replication than the two intramuscular doses of the adjuvanted HMPV pre-F subunit antigens. B/HPIV3 expressing a codon-optimized pre-fusion stabilized HMPV F was advanced to a pediatric clinical study.