FluB-RAM and FluB-RANS: Genome Rearrangement as Safe and Efficacious Live Attenuated Influenza B Virus Vaccines

Abstract

Influenza B virus (IBV) is considered a major respiratory pathogen responsible for seasonal respiratory disease in humans, particularly severe in children and the elderly. Seasonal influenza vaccination is considered the most efficient strategy to prevent and control IBV infections. Live attenuated influenza virus vaccines (LAIVs) are thought to induce both humoral and cellular immune responses by mimicking a natural infection, but their effectiveness has recently come into question. Thus, the opportunity exists to find alternative approaches to improve overall influenza vaccine effectiveness. Two alternative IBV backbones were developed with rearranged genomes, rearranged M (FluB-RAM) and a rearranged NS (FluB-RANS). Both rearranged viruses showed temperature sensitivity in vitro compared with the WT type B/Bris strain, were genetically stable over multiple passages in embryonated chicken eggs and were attenuated in vivo in mice. In a prime-boost regime in naïve mice, both rearranged viruses induced antibodies against HA with hemagglutination inhibition titers considered of protective value. In addition, antibodies against NA and NP were readily detected with potential protective value. Upon lethal IBV challenge, mice previously vaccinated with either FluB-RAM or FluB-RANS were completely protected against clinical disease and mortality. In conclusion, genome re-arrangement renders efficacious LAIV candidates to protect mice against IBV.

FLUAV RAM-IGIP: A modified live influenza virus vaccine that enhances humoral and mucosal responses against influenza

“…The expected amplicon sizes for PB1-M2 is 679 bp and for PB1 WT is 273 bp. This observation aligns with a similar strategy for FLUBV (6). Consistent with a previous report (12), the inclusion of IGIP in the HA remained stable (Fig 1D).…”

“…The FLUAV RAM attenuating strategy involves inserting the M2 ion channel ORF downstream of the PB1 ORF (segment 2) and separating them with a spacer encoding the Thosea asigna virus 2A protease (2ATaV), whose activity is predicted to release M2 from PB1 during translation. Multiple early stop codons are introduced in segment 7 to eliminate M2 expression(6). We evaluated the polymerase activity mediated by the RAM PB1-M2 gene derived from the A/California/04/2009 (H1N1) (Ca/04) strain(14) in a minigenome assay at different temperatures (Fig S1) and compared it to the previously described platform with temperature sensitive mutations (att)(15).…”

“…Viruses were titrated by tissue culture infectious dose 50 (TCID50), and virus titers were established by the Reed and Muench method (51). Virus sequences were confirmed by next-generation sequencing and Sanger sequencing, as previously described (6, 52)…”

“…FLUAV and FLUBV viruses are amenable to substantial genome rearrangements, with the PB1 segment tolerating the addition of various foreign sequences, specifically M2 (RAM) and NEP (RANS) (5, 6). Modifications in segment 7 to prevent expression of M2 in RAM or in segment 8 to prevent expression of NEP in RANS further attenuate these viruses (5, 6). FLUAV and FLUBV with RAM or RANS rearrangements induced protection against influenza virus challenge in different mammalian models (5, 6).…”

“…Modifications in segment 7 to prevent expression of M2 in RAM or in segment 8 to prevent expression of NEP in RANS further attenuate these viruses (5, 6). FLUAV and FLUBV with RAM or RANS rearrangements induced protection against influenza virus challenge in different mammalian models (5, 6). Unlike current LAIVs, the genome rearrangement MLV strategy can be used to update the entire vaccine virus backbone.…”