Live Attenuated Influenza Virus as a Vector for Multivalent T-Cell Vaccines: Targeting RSV, hMPV, and PIV3

Background/Objectives: Respiratory syncytial virus (RSV), human metapneumovirus (hMPV), and parainfluenza virus type 3 (PIV3) are leading causes of acute respiratory infections in children and the elderly, yet no licensed T-cell vaccines are available. This study aimed to develop multivalent T-cell vaccine candidates against these pathogens using a live attenuated influenza virus (LAIV) vector platform. Methods: Conserved F, N, and M proteins of RSV, hMPV, and PIV3 were identified through multiple sequence alignments. Fragments enriched with experimentally confirmed and predicted T-cell epitopes were selected using the IEDB and NetMHCpan servers. These fragments were assembled into polyepitope immunogenic cassettes, and their selected order was determined by thermodynamic analysis of mRNA secondary structures using the RNAfold Web Server. The selected cassettes were cloned into the neuraminidase (NA) gene of a cold-adapted LAIV vector. Recombinant viruses were rescued by reverse genetics and assessed for replicative fitness in embryonated chicken eggs and MDCK cells, NA enzymatic activity and genetic stability upon serial passaging. Results: Four cassettes were designed for RSV, three for hMPV, and one for PIV3, all containing fragments with multiple T-cell epitopes. Three recombinant viruses of LAIV/RSV type and three of LAIV/hMPV type were successfully rescued, while attempts to recover the remaining recombinant viruses, i.e., LAIV/RSV and LAIV/PIV3, were not successful. All rescued recombinant viruses replicated to titers comparable to the parental LAIV strain and retained the full-length insert for at least eight passages in eggs. Importantly, NA enzymatic activity of the LAIV vector was not compromised by the insertion of the polyepitope T-cell cassettes. Conclusions: We developed a panel of recombinant T cell-based vaccine candidates against RSV and hMPV using the LAIV vector platform. These recombinant viruses encode conserved T-cell epitopes of the target viruses while retaining the biological properties of LAIV strains. Taken together, these characteristics warrant further evaluation of these recombinant viruses in appropriate relevant in vitro models to directly assess their immunogenicity in terms of stimulating a T-cell response against target pathogens.