Amino acid 138 in the HA of a H3N2 subtype influenza A virus increases affinity for the lower respiratory tract and alveolar macrophages in pigs.

Influenza A virus (FLUAV) infects a wide range of hosts and human-to-swine spillover events are frequently reported. However, only a few of these human viruses have become established in pigs and the host barriers and molecular mechanisms driving adaptation to the swine host remain poorly understood. We previously found that infection of pigs with a 2:6 reassortant virus (hVIC/11) containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from the human strain A/Victoria/361/2011 (H3N2) and internal gene segments of an endemic swine strain (sOH/04) resulted in a fixed amino acid substitution in the HA (A138S, mature H3 HA numbering). In silico analysis revealed that S138 became predominant among swine H3N2 virus sequences deposited in public databases, while 138A predominates in human isolates. To understand the role of the HA A138S substitution in the adaptation of a human-origin FLUAV HA to swine, we infected pigs with the hVIC/11A138S mutant and analyzed pathogenesis and transmission compared to hVIC/11 and sOH/04. Our results showed that the hVIC/11A138S virus had an intermediary pathogenesis between hVIC/11 and sOH/04. The hVIC/11A138S infected the upper respiratory tract, right caudal, and both cranial lobes while hVIC/11 was only detected in nose and trachea samples. Viruses induced a distinct expression pattern of various pro-inflammatory cytokines such as IL-8, TNF-α, and IFN-ß. Flow cytometric analysis of lung samples revealed a significant reduction of porcine alveolar macrophages (PAMs) in hVIC/11A138S-infected pigs compared to hVIC/11 while a MHCIIlowCD163neg population was increased. The hVIC/11A138S showed a higher affinity for PAMs than hVIC/11, noted as an increase of infected PAMs in bronchoalveolar lavage fluid (BALF), and showed no differences in the percentage of HA-positive PAMs compared to sOH/04. This increased infection of PAMs led to an increase of granulocyte-monocyte colony-stimulating factor (GM-CSF) stimulation but a reduced expression of peroxisome proliferator-activated receptor gamma (PPARγ) in the sOH/04-infected group. Analysis using the PAM cell line 3D4/21 revealed that the A138S substitution improved replication and apoptosis induction in this cell type compared to hVIC/11 but at lower levels than sOH/04. Overall, our study indicates that adaptation of human viruses to the swine host involves an increased affinity for the lower respiratory tract and alveolar macrophages.

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

Current influenza A vaccines fall short, leaving both humans and animals vulnerable. To address this issue, we have developed attenuated modified live virus (MLV) vaccines against influenza using genome rearrangement techniques targeting the internal gene segments of FLUAV. The rearranged M2 (RAM) strategy involves cloning the M2 ORF downstream of the PB1 ORF in segment 2 and incorporating multiple early stop codons within the M2 ORF in segment 7. Additionally, the IgA-inducing protein (IGIP) coding region was inserted into the HA segment to further attenuate the virus and enhance protective mucosal responses. RAM-IGIP viruses exhibit similar growth rates to wild type (WT) viruses in vitro and remain stable during multiple passages in cells and embryonated eggs. The safety, immunogenicity, and protective efficacy of the RAM-IGIP MLV vaccine against the prototypical 2009 pandemic H1N1 strain A/California/04/2009 (H1N1) (Ca/04) were evaluated in Balb/c mice and compared to a prototypic cold-adapted live attenuated virus vaccine. The results demonstrate that the RAM-IGIP virus exhibits attenuated virulence in vivo. Mice vaccinated with RAM-IGIP and subsequently challenged with an aggressive lethal dose of the Ca/04 strain exhibited complete protection. Analysis of the humoral immune response revealed that the inclusion of IGIP enhanced the production of neutralizing antibodies and augmented the antibody-dependent cellular cytotoxicity response. Similarly, the RAM-IGIP potentiated the mucosal immune response against various FLUAV subtypes. Moreover, increased antibodies against NP and NA responses were observed. These findings support the development of MLVs utilizing genome rearrangement strategies in conjunction with the incorporation of immunomodulators. IMPORTANCE: Current influenza vaccines offer suboptimal protection, leaving both humans and animals vulnerable. Our novel attenuated MLV vaccine, built by rearranging FLUAV genome segments and incorporating the IgA-inducing protein, shows promising results. This RAM-IGIP vaccine exhibits safe attenuation, robust immune responses, and complete protection against lethal viral challenge in mice. Its ability to stimulate broad-spectrum humoral and mucosal immunity against diverse FLUAV subtypes makes it a highly promising candidate for improved influenza vaccines.

Use of Reverse Genetics for the Generation of Recombinant Influenza Viruses Carrying Nanoluciferase.

Influenza A (FLUAV) and influenza B (FLUBV) viruses are human and/or animal pathogens widely studied due to their importance to public health and animal production. Both FLUAV and FLUBV possess a genome composed of eight viral gene segments. For reverse genetics of influenza viruses, transcription of the mRNA for the viral proteins is typically done from a plasmid encoding an RNA polymerase II (pol II) promoter element upstream of cloned viral cDNA and expressed like host mRNA. On the other side, the synthesis of the negative-sense, single-stranded, uncapped vRNAs can be accomplished by the host's RNA polymerase I (pol I). The reverse genetics for influenza has allowed the manipulation of influenza genomes incorporating heterogeneous sequences into different segments of the influenza genome, such as reporter genes. In this chapter, we outline the protocol from the generation of reverse genetic plasmid that can be applied for the cloning of any of the segments of FLUAV or FLUBV. Furthermore, we describe a protocol for generating FLUAV or FLUBV recombinant viruses carrying Nanoluciferase (NLuc) in the PB1 gene using reverse genetics. Finally, we delineate a microneutralization protocol using FLUAV-NLuc or FLUBV-NLuc viruses optimized for the use of antibodies from different sources (mice, ferrets, avian, etc.), which provides a more sensitive, reliable, and avidity-independent method to assess the presence of neutralizing antibodies against FLUAV or FLUBV.