Pathobiology of an NS1-Truncated H3N2 Swine Influenza Virus Strain in Pigs.

Virus strains in the live attenuated influenza vaccine (LAIV) for swine in the United States that was on the market until 2020 encode a truncated nonstructural protein 1 of 126 amino acids (NS1del126). Their attenuation is believed to be due to an impaired ability to counteract the type I interferon (IFN)-mediated antiviral host response. However, this mechanism has been documented only in vitro for H3N2 strain A/swine/Texas/4199-2/98 NS1del126 (lvTX98), and several cases of clinical respiratory disease in the field were associated with the LAIV strains. We therefore further examined the pathobiology, including type I IFN induction, of lvTX98 in pigs and compared it with IFN induction in pig kidney-15 (PK-15) cells. lvTX98 induced up to 3-fold-higher type I IFN titers than wild-type TX98 (wtTX98) after inoculation of PK-15 cells at a high multiplicity of infection, while virus replication kinetics were similar. Mean nasal lvTX98 excretion by intranasally inoculated pigs was on average 50 times lower than that for wtTX98 but still reached titers of up to 4.3 log10 50% tissue culture infective doses/mL. After intratracheal inoculation, mean lvTX98 titers in the lower respiratory tract were significantly reduced at 18 to 48 h postinoculation (hpi) but similar to wtTX98 titers at 72 hpi. lvTX98 caused milder clinical signs than wtTX98 but induced comparable levels of microscopic and macroscopic lung lesions, peak neutrophil infiltration, and peak type I IFN. Thus, lvTX98 was partly attenuated in pigs, but this could not be associated with higher type I IFN levels. IMPORTANCE Swine influenza A viruses (swIAVs) with a truncated NS1del126 protein were strongly attenuated in previous laboratory-based safety studies and therefore approved for use as LAIVs for swine in the United States. In the field, however, the LAIV strains were detected in diagnostic samples and could regain a wild-type NS1 via reassortment with endemic swIAVs. This suggests a significant degree of LAIV replication and urges further investigation of the level and mechanism of attenuation of these LAIV strains in vivo. Here, we show that H3N2 LAIV strain lvTX98 is only partly attenuated in pigs and is excreted at significant titers after intranasal vaccination. Attenuation and restricted replication of lvTX98 in vivo seemed to be associated with the loss of NS1 functions other than type I IFN antagonism. Our findings can help to explain the occurrence of clinical respiratory disease and reassortment events associated with NS1del126-based LAIV strains in the field.

Efficacy of the NS1-truncated live attenuated influenza virus vaccine for swine against infection with viruses of major North American and European H3N2 lineages.

Control of swine influenza A virus (swIAV) in North America and Europe is complicated because multiple antigenically distinct swIAV strains co-circulate in the field, and no vaccine is available that can provide broad cross-protection against all these swIAVs. In 2017, the first live attenuated influenza vaccine (LAIV) for swine was licensed in the US. The non-structural protein 1 (NS1)-truncated cluster I H3N2 strain A/swine/Texas/4199-2/98 NS1del126 (TX98 LAIV) in this vaccine provides partial cross-protection against heterologous North American cluster II and IV H3N2 swIAV strains. Its efficacy against European or more recent North American H3N2 lineages remains to be investigated. In this study, we evaluated the level of cross-protection against heterologous IAVs representative of the major H3N2 swIAV lineages in Europe and North America. TX98 LAIV prevented both nasal shedding and replication in the lungs of a North American cluster IV H3N2 swIAV for 2/4 pigs, prevented considerable nasal shedding of a North American novel human-like H3N2 swIAV for 2/4 pigs, and reduced replication of a European H3N2 swIAV in the lower respiratory tract to minimal titers for 1/3 pigs. Although TX98 LAIV elicited neutralizing antibodies against the homologous virus in serum and to a lesser extent in nose and lungs, no significant cross-reactive antibody titers against the heterologous swIAVs were detected. Partial cross-protection therefore likely relies on cellular and mucosal immune responses against conserved parts of the swIAV proteins. Since TX98 LAIV can offer partial protection against a broad range of H3N2 swIAVs, it might be a suitable priming vaccine for use in a heterologous prime-boost vaccination strategy.

Genetic and antigenic evolution of H1 swine influenza A viruses isolated in Belgium and the Netherlands from 2014 through 2019.

Surveillance of swine influenza A viruses (swIAV) allows timely detection and identification of new variants with potential zoonotic risks. In this study, we aimed to identify swIAV subtypes that circulated in pigs in Belgium and the Netherlands between 2014 and 2019, and characterize their genetic and antigenic evolution. We subtyped all isolates and analyzed hemagglutinin sequences and hemagglutination inhibition assay data for H1 swIAV, which were the dominant HA subtype. We also analyzed whole genome sequences (WGS) of selected isolates. Out of 200 samples, 89 tested positive for swIAV. swIAV of H1N1, H1N2 and H3N2 subtypes were detected. Analysis of WGS of 18 H1 swIAV isolates revealed three newly emerged genotypes. The European avian-like H1 swIAV (lineage 1C) were predominant and accounted for 47.2% of the total isolates. They were shown to evolve faster than the European human-like H1 (1B lineage) swIAV, which represented 27% of the isolates. The 2009 pandemic H1 swIAV (lineage 1A) accounted for only 5.6% of the isolates and showed divergence from their precursor virus. These results point to the increasing divergence of swIAV and stress the need for continuous surveillance of swIAV.

Efficacy of Heterologous Prime-Boost Vaccination with H3N2 Influenza Viruses in Pre-Immune Individuals: Studies in the Pig Model.

In a previous study in influenza-naïve pigs, heterologous prime-boost vaccination with monovalent, adjuvanted whole inactivated vaccines (WIV) based on the European swine influenza A virus (SwIAV) strain, A/swine/Gent/172/2008 (G08), followed by the US SwIAV strain, A/swine/Pennsylvania/A01076777/2010 (PA10), was shown to induce broadly cross-reactive hemagglutination inhibition (HI) antibodies against 12 out of 15 antigenically distinct H3N2 influenza strains. Here, we used the pig model to examine the efficacy of that particular heterologous prime-boost vaccination regimen, in individuals with pre-existing infection-immunity. Pigs were first inoculated intranasally with the human H3N2 strain, A/Nanchang/933/1995. Seven weeks later, they were vaccinated intramuscularly with G08 followed by PA10 or vice versa. We examined serum antibody responses against the hemagglutinin and neuraminidase, and antibody-secreting cell (ASC) responses in peripheral blood, draining lymph nodes, and nasal mucosa (NMC), in ELISPOT assays. Vaccination induced up to 10-fold higher HI antibody titers than in naïve pigs, with broader cross-reactivity, and protection against challenge with an antigenically distant H3N2 strain. It also boosted ASC responses in lymph nodes and NMC. Our results show that intramuscular administration of WIV can lead to enhanced antibody responses and cross-reactivity in pre-immune subjects, and recall of ASC responses in lymph nodes and NMC.