Surveillance and Genomic Characterization of Influenza A and D Viruses in Swine, Belgium and the Netherlands, 2019-2021.

During 2019-2021, we isolated 62 swine influenza A viruses in Belgium and the Netherlands. We also detected influenza D in pigs in the Netherlands. The ever-changing diversity of influenza viruses and the identification of influenza D emphasize the need for more virus surveillance.

Human Immunity and Susceptibility to Influenza A(H3) Viruses of Avian, Equine, and Swine Origin.

Influenza A viruses (IAVs) of subtype H3 that infect humans are antigenically divergent from those of birds, horses, and swine. Human immunity against these viruses might be limited, implying potential pandemic risk. To determine human risk, we selected 4 avian, 1 equine, and 3 swine IAVs representing major H3 lineages. We tested serum collected during 2017-2018 from 286 persons in Belgium for hemagglutination inhibiting antibodies and virus neutralizing antibodies against those animal-origin IAVs and tested replication in human airway epithelia. Seroprevalence rates for circulating IAVs from swine in North America were >51%, swine in Europe 7%-37%, and birds and equids ≤12%. Replication was efficient for cluster IV-A IAVs from swine in North America and IAVs from swine in Europe, intermediate for IAVs from horses and poultry, and absent for IAVs from wild birds and a novel human-like swine IAV in North America. Public health risk may be highest for swine H3 IAVs.

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.

Infectivity and transmissibility of an avian H3N1 influenza virus in pigs.

In 2019 a low pathogenic H3N1 avian influenza virus (AIV) caused an outbreak in Belgian poultry farms, characterized by an unusually high mortality in chickens. Influenza A viruses of the H1 and H3 subtype can infect pigs and become established in swine populations. Therefore, the H3N1 epizootic raised concern about AIV transmission to pigs and from pigs to humans. Here, we assessed the replication efficiency of this virus in explants of the porcine respiratory tract and in pigs, using virus titration and/or RT-qPCR. We also examined transmission from directly, intranasally inoculated pigs to contact pigs. The H3N1 AIV replicated to moderate titers in explants of the bronchioles and lungs, but not in the nasal mucosa or trachea. In the pig infection study, infectious virus was only detected in a few lung samples collected between 1 and 3 days post-inoculation. Virus titers were between 1.7 and 4.8 log10 TCID50. In line with the ex vivo experiment, no virus was isolated from the upper respiratory tract of pigs. In the transmission experiment, we could not detect virus transmission from directly inoculated to contact pigs. An increase in serum antibody titers was observed only in the inoculated pigs. We conclude that the porcine respiratory tract tissue explants can be a useful tool to assess the replication efficiency of AIVs in pigs. The H3N1 AIV examined here is unlikely to pose a risk to swine populations. However, continuous risk assessment studies of emerging AIVs in pigs are necessary, since different virus strains will have different genotypic and phenotypic traits.

Alternating 3 different influenza vaccines for swine in Europe for a broader antibody response and protection.

Heterologous prime-boost vaccination with experimental or commercial influenza vaccines has been successful in various animal species. In this study, we have examined the efficacy of alternating 3 different European commercial swine influenza A virus (swIAV) vaccines: the trivalent Respiporc® FLU3 (TIV), the bivalent GRIPORK® (BIV) and the monovalent Respiporc® FLUpan H1N1 (MOV). Five groups of 6 pigs each received 3 vaccinations at 4-6 week intervals in a homologous or heterologous prime-boost regimen. A sixth group served as a mock-vaccinated challenge control. Four weeks after the last vaccination, pigs were challenged intranasally with a European avian-like H1N1 (1C.2.1) swIAV, which was antigenically distinct from the vaccine strains. One heterologous prime-boost group (TIV-BIV-MOV) had higher hemagglutination inhibition (HI) and neuraminidase inhibition antibody responses against a panel of antigenically distinct H1N1, H1N2 and H3N2 IAVs than the other heterologous prime-boost group (BIV-TIV-MOV) and the homologous prime-boost groups (3xTIV; 3xBIV; 3xMOV). Group TIV-BIV-MOV had seroprotective HI titers (≥ 40) against 56% of the tested viruses compared to 33% in group BIV-TIV-MOV and 22-39% in the homologous prime-boost groups. Post-challenge, group TIV-BIV-MOV was the single group with significantly reduced virus titers in all respiratory samples compared to the challenge control group. Our results suggest that the use of different commercial swIAV vaccines for successive vaccinations may result in broader antibody responses and protection than the traditional, homologous prime-boost vaccination regimens. In addition, the order in which the different vaccines are administered seems to affect the breadth of the antibody response and protection.

Human Infection with Eurasian Avian-Like Swine Influenza A(H1N1) Virus, the Netherlands, September 2019.

We report a zoonotic infection of a pig farmer in the Netherlands with a Eurasian avian-like swine influenza A(H1N1) virus that was also detected in the farmed pigs. Both viruses were antigenically and genetically characterized. Continued surveillance of swine influenza A viruses is needed for risk assessment in humans and swine.

Detection of H1 Swine Influenza A Virus Antibodies in Human Serum Samples by Age Group1.

Most H1 influenza A viruses (IAVs) of swine are derived from past human viruses. As human population immunity against these IAVs gradually decreases, the risk of reintroduction to humans increases. We examined 549 serum samples from persons 0-97 years of age collected in Belgium during 2017-2018 for hemagglutination inhibiting and virus neutralizing antibodies against 7 major H1 swine IAV (swIAV) clades and 3 human progenitor IAVs. Seroprevalence (titers >40) rates were >50% for classical swine and European human-like swIAVs, >24% for North American human-like δ1a and Asian avian-like swIAVs, and <10% for North American human-like δ1b and European avian-like swIAVs, but rates were age-dependent. Antibody titers against human-like swIAVs and supposed human precursor IAVs correlated with correlation coefficients of 0.30-0.86. Our serologic findings suggest that European avian-like, clade 1C.2.1, and North American human-like δ1b, clade 1B.2.2.2, H1 swIAVs pose the highest pandemic risk.

Role of Substitutions in the Hemagglutinin in the Emergence of the 1968 Pandemic Influenza Virus.

Hemagglutinin (HA) of H3N2/1968 pandemic influenza viruses differs from the putative avian precursor by seven amino acid substitutions. Substitutions Q226L and G228S are known to be essential for adaptation of avian HA to mammals. We found that introduction of avian-virus-like amino acids at five other HA positions (positions 62, 81, 92, 144, and 193) of A/Hong Kong/1/1968 virus decreased viral replication in human cells and transmission in pigs. Thus, substitutions at some of these positions facilitated emergence of the pandemic virus.

Molecular Epidemiology and Evolution of Influenza Viruses Circulating within European Swine between 2009 and 2013.

UNLABELLED: The emergence in humans of the A(H1N1)pdm09 influenza virus, a complex reassortant virus of swine origin, highlighted the importance of worldwide influenza virus surveillance in swine. To date, large-scale surveillance studies have been reported for southern China and North America, but such data have not yet been described for Europe. We report the first large-scale genomic characterization of 290 swine influenza viruses collected from 14 European countries between 2009 and 2013. A total of 23 distinct genotypes were identified, with the 7 most common comprising 82% of the incidence. Contrasting epidemiological dynamics were observed for two of these genotypes, H1huN2 and H3N2, with the former showing multiple long-lived geographically isolated lineages, while the latter had short-lived geographically diffuse lineages. At least 32 human-swine transmission events have resulted in A(H1N1)pdm09 becoming established at a mean frequency of 8% across European countries. Notably, swine in the United Kingdom have largely had a replacement of the endemic Eurasian avian virus-like ("avian-like") genotypes with A(H1N1)pdm09-derived genotypes. The high number of reassortant genotypes observed in European swine, combined with the identification of a genotype similar to the A(H3N2)v genotype in North America, underlines the importance of continued swine surveillance in Europe for the purposes of maintaining public health. This report further reveals that the emergences and drivers of virus evolution in swine differ at the global level. IMPORTANCE: The influenza A(H1N1)pdm09 virus contains a reassortant genome with segments derived from separate virus lineages that evolved in different regions of the world. In particular, its neuraminidase and matrix segments were derived from the Eurasian avian virus-like ("avian-like") lineage that emerged in European swine in the 1970s. However, while large-scale genomic characterization of swine has been reported for southern China and North America, no equivalent study has yet been reported for Europe. Surveillance of swine herds across Europe between 2009 and 2013 revealed that the A(H1N1)pdm09 virus is established in European swine, increasing the number of circulating lineages in the region and increasing the possibility of the emergence of a genotype with human pandemic potential. It also has implications for veterinary health, making prevention through vaccination more challenging. The identification of a genotype similar to the A(H3N2)v genotype, causing zoonoses at North American agricultural fairs, underlines the importance of continued genomic characterization in European swine.

Cross-protection against European swine influenza viruses in the context of infection immunity against the 2009 pandemic H1N1 virus: studies in the pig model of influenza.

Pigs are natural hosts for the same influenza virus subtypes as humans and are a valuable model for cross-protection studies with influenza. In this study, we have used the pig model to examine the extent of virological protection between a) the 2009 pandemic H1N1 (pH1N1) virus and three different European H1 swine influenza virus (SIV) lineages, and b) these H1 viruses and a European H3N2 SIV. Pigs were inoculated intranasally with representative strains of each virus lineage with 6- and 17-week intervals between H1 inoculations and between H1 and H3 inoculations, respectively. Virus titers in nasal swabs and/or tissues of the respiratory tract were determined after each inoculation. There was substantial though differing cross-protection between pH1N1 and other H1 viruses, which was directly correlated with the relatedness in the viral hemagglutinin (HA) and neuraminidase (NA) proteins. Cross-protection against H3N2 was almost complete in pigs with immunity against H1N2, but was weak in H1N1/pH1N1-immune pigs. In conclusion, infection with a live, wild type influenza virus may offer substantial cross-lineage protection against viruses of the same HA and/or NA subtype. True heterosubtypic protection, in contrast, appears to be minimal in natural influenza virus hosts. We discuss our findings in the light of the zoonotic and pandemic risks of SIVs.

Swine influenza virus vaccines: to change or not to change-that's the question.

Commercial vaccines currently available against swine influenza virus (SIV) are inactivated, adjuvanted, whole virus vaccines, based on H1N1 and/or H3N2 and/or H1N2 SIVs. In keeping with the antigenic and genetic differences between SIVs circulating in Europe and the US, the vaccines for each region are produced locally and contain different strains. Even within a continent, there is no standardization of vaccine strains, and the antigen mass and adjuvants can also differ between different commercial products. Recombinant protein vaccines against SIV, vector, and DNA vaccines, and vaccines attenuated by reverse genetics have been tested in experimental studies, but they have not yet reached the market. In this review, we aim to present a critical analysis of the performance of commercial inactivated and novel generation SIV vaccines in experimental vaccination challenge studies in pigs. We pay special attention to the differences between commercial SIV vaccines and vaccination attitudes in Europe and in North America, to the issue of vaccine strain selection and changes, and to the potential advantages of novel generation vaccines over the traditional killed SIV vaccines.

Sequential vaccinations with divergent H1N1 influenza virus strains induce multi-H1 clade neutralizing antibodies in swine.

Vaccines that protect against any H1N1 influenza A virus strain would be advantageous for use in pigs and humans. Here, we try to induce a pan-H1N1 antibody response in pigs by sequential vaccination with antigenically divergent H1N1 strains. Adjuvanted whole inactivated vaccines are given intramuscularly in various two- and three-dose regimens. Three doses of heterologous monovalent H1N1 vaccine result in seroprotective neutralizing antibodies against 71% of a diverse panel of human and swine H1 strains, detectable antibodies against 88% of strains, and sterile cross-clade immunity against two heterologous challenge strains. This strategy outperforms any two-dose regimen and is as good or better than giving three doses of matched trivalent vaccine. Neutralizing antibodies are H1-specific, and the second heterologous booster enhances reactivity with conserved epitopes in the HA head. We show that even the most traditional influenza vaccines can offer surprisingly broad protection if they are administered in an alternative way.

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.

Exploring Prime-Boost Vaccination Regimens with Different H1N1 Swine Influenza A Virus Strains and Vaccine Platforms.

In a previous vaccination study in pigs, heterologous prime-boost vaccination with whole-inactivated H1N1 virus vaccines (WIV) induced superior antibody responses and protection compared to homologous prime-boost vaccination. However, no pan-H1 antibody response was induced. Therefore, to stimulate both local and systemic immune responses, we first vaccinated pigs intranasally with a pseudorabies vector vaccine expressing the pH1N1 hemagglutinin (prvCA09) followed by a homologous or heterologous WIV booster vaccine. Homologous and heterologous WIV-WIV vaccinated groups and mock-vaccinated or prvCA09 single-vaccinated pigs served as control groups. Five weeks after the second vaccination, pigs were challenged with a homologous pH1N1 or one of two heterologous H1N2 swine influenza A virus strains. A single prvCA09 vaccination resulted in complete protection against homologous challenge, and vector-WIV vaccinated groups were significantly better protected against heterologous challenge compared to the challenge control group or WIV-WIV vaccinated groups. Furthermore, vector-WIV vaccination resulted in broader hemagglutination inhibition antibody responses compared to WIV-WIV vaccination and higher numbers of antibody-secreting cells in peripheral blood, draining lymph nodes and nasal mucosa. However, even though vector-WIV vaccination induced stronger antibody responses and protection, we still failed to induce a pan-H1 antibody response.

Swine influenza virus antibodies in humans, western Europe, 2009.

Serologic studies for swine influenza viruses (SIVs) in humans with occupational exposure to swine have been reported from the Americas but not from Europe. We compared levels of neutralizing antibodies against 3 influenza viruses--pandemic (H1N1) 2009, an avian-like enzootic subtype H1N1 SIV, and a 2007-08 seasonal subtype H1N1--in 211 persons with swine contact and 224 matched controls in Luxembourg. Persons whose profession involved contact with swine had more neutralizing antibodies against SIV and pandemic (H1N1) 2009 virus than did the controls. Controls also had antibodies against these viruses although exposure to them was unlikely. Antibodies against SIV and pandemic (H1N1) 2009 virus correlated with each other but not with seasonal subtype H1N1 virus. Sequential exposure to variants of seasonal influenza (H1N1) viruses may have increased chances for serologic cross-reactivity with antigenically distinct viruses. Further studies are needed to determine the extent to which serologic responses correlate with infection.

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.

Ancestral sequence reconstruction pinpoints adaptations that enable avian influenza virus transmission in pigs.

Understanding the evolutionary adaptations that enable avian influenza viruses to transmit in mammalian hosts could allow better detection of zoonotic viruses with pandemic potential. We applied ancestral sequence reconstruction to gain viruses representing different adaptive stages of the European avian-like (EA) H1N1 swine influenza virus as it transitioned from avian to swine hosts since 1979. Ancestral viruses representing the avian-like precursor virus and EA swine influenza viruses from 1979-1983, 1984-1987 and 1988-1992 were reconstructed and characterized. Glycan-binding analyses showed stepwise changes in the haemagglutinin receptor-binding specificity of the EA swine influenza viruses-that is, from recognition of both α2,3- and α2,6-linked sialosides to recognition of α2,6-linked sialosides only; however, efficient transmission in piglets was enabled by adaptive changes in the viral polymerase protein and nucleoprotein, which have been fixed since 1983. PB1-Q621R and NP-R351K increased viral replication and transmission in piglets when introduced into the 1979-1983 ancestral virus that lacked efficient transmissibility. The stepwise adaptation of an avian influenza virus to a mammalian host suggests that there may be opportunities to intervene and prevent interspecies jumps through strategic coordination of surveillance and risk assessment activities.

Replication of avian, human and swine influenza viruses in porcine respiratory explants and association with sialic acid distribution.

BACKGROUND: Throughout the history of human influenza pandemics, pigs have been considered the most likely "mixing vessel" for reassortment between human and avian influenza viruses (AIVs). However, the replication efficiencies of influenza viruses from various hosts, as well as the expression of sialic acid (Sia) receptor variants in the entire porcine respiratory tract have never been studied in detail. Therefore, we established porcine nasal, tracheal, bronchial and lung explants, which cover the entire porcine respiratory tract with maximal similarity to the in vivo situation. Subsequently, we assessed virus yields of three porcine, two human and six AIVs in these explants. Since our results on virus replication were in disagreement with the previously reported presence of putative avian virus receptors in the trachea, we additionally studied the distribution of sialic acid receptors by means of lectin histochemistry. Human (Sia alpha2-6Gal) and avian virus receptors (Sia alpha2-3Gal) were identified with Sambucus Nigra and Maackia amurensis lectins respectively. RESULTS: Compared to swine and human influenza viruses, replication of the AIVs was limited in all cultures but most strikingly in nasal and tracheal explants. Results of virus titrations were confirmed by quantification of infected cells using immunohistochemistry. By lectin histochemistry we found moderate to abundant expression of the human-like virus receptors in all explant systems but minimal binding of the lectins that identify avian-like receptors, especially in the nasal, tracheal and bronchial epithelium. CONCLUSIONS: The species barrier that restricts the transmission of influenza viruses from one host to another remains preserved in our porcine respiratory explants. Therefore this system offers a valuable alternative to study virus and/or host properties required for adaptation or reassortment of influenza viruses. Our results indicate that, based on the expression of Sia receptors alone, the pig is unlikely to be a more appropriate mixing vessel for influenza viruses than humans. We conclude that too little is known on the exact mechanism and on predisposing factors for reassortment to assess the true role of the pig in the emergence of novel influenza viruses.

Efficient transmission of swine-adapted but not wholly avian influenza viruses among pigs and from pigs to ferrets.

BACKGROUND: Pigs are considered to be intermediate hosts for the transmission of avian influenza viruses (AIVs) between birds and humans, but the transmissibility of AIVs among pigs and from pigs to other mammals remains largely unexplored. METHODS: In an initial study, we confirmed the replication potential of various low-pathogenic AIVs in pigs and ferrets, which we used as a model for humans. We then examined the transmissibility of 6 selected low-pathogenic AIVs among pigs (by direct contact) and from pigs to ferrets (by air contact). RESULTS: Although H1N1 and H3N2 swine influenza viruses spread readily from inoculated pigs to contact pigs and ferrets, evidence for AIV transmission was extremely rare. The fact that infected pigs shed lower amounts of AIVs than of swine influenza viruses may contribute to the reduced transmissibility of AIVs. CONCLUSIONS: Our data indicate that swine-adapted influenza viruses spread readily among pigs and from pigs to other susceptible mammalians and support the notion that AIVs undergo genetic adaptation to efficiently cross the species barrier. Our transmission models hold potential to study the factors that lead to the generation of pandemic influenza viruses.

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.