An RNA replicon vaccine encoding HA and NA prevents shedding of antigen- drifted 2009 pandemic H1N1 influenza virus in the pig model

An RNA replicon vaccine encoding HA and NA prevents shedding of antigen- drifted 2009 pandemic H1N1 influenza virus in the pig model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article An RNA replicon vaccine encoding HA and NA prevents shedding of antigen- drifted 2009 pandemic H1N1 influenza virus in the pig model Obdulio Garcia-Nicolas, Lisa Butticaz, Robin Avanthay, Nicolas Ruggli, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7818522/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Mar, 2026 Read the published version in npj Vaccines → Version 1 posted 10 You are reading this latest preprint version Abstract Seasonal influenza viruses escape the human immune response by antigenic drift, i.e. the positive selection of point mutations that prevent the binding of inhibitory antibodies to the influenza antigens HA and NA. The efficacy of seasonal influenza vaccines can be less than 50% if the selected influenza vaccine strain does not match the antigenic characteristics of the circulating seasonal influenza virus. In this study, we used the porcine model to evaluate the efficacy of an RNA replicon vaccine encoding the HA and NA antigens of A/Hamburg/4/2009 (H1N1) (H1N1 HH4/09 ) in inducing cross-reactive immunity. We found that a single intramuscular immunization with this vaccine elicited high levels of antibodies with H1N1 HH4/09 -neutralizing activity and potent N1-sialidase inhibition. A second immunization with the same H1/N1 RNA replicon particles or with a live-attenuated influenza vaccine (LAIV) based on a modified H1N1 HH4/09 virus boosted the inhibitory activity of the immune sera against the antigen-drifted A/Victoria/2570/2019 (H1N1) (H1N1 Vic/19 ) strain. Interestingly, vaccination elicited N1-specific antibodies that also inhibited the activity of avian N1 sialidase and potently inhibited the replication of A/cattle/Texas/063224-24-1/2024 (H5N1) (H5N1 Tex/24 ) in vitro . When challenged nasally with a H1N1 HH4/09 /H1N1 Vic/19 6:2 reassortant virus encoding the HA and NA antigens of H1N1 Vic/19 , immunized pigs did not shed infectious virus while the control animals did, suggesting that homologous prime/boost vaccination with H1/N1 replicon particles can block virus replication in the upper respiratory tract as efficiently as the heterologous RNA replicon prime/LAIV boost immunization regimen. In conclusion, RNA replicons encoding both HA and NA either used alone or in combination with LAIV mediate protection against antigen-drifted influenza viruses and reduce the risk of vaccination breakthroughs due to antigen mismatch. Furthermore, this vaccine may also limit the infection by zoonotic H5N1 viruses. Biological sciences/Immunology Biological sciences/Microbiology Neuraminidase hemagglutinin RNA replicon particle virus shedding 2009 pandemic H1N1 influenza virus H5N1 highly pathogenic avian influenza virus prime-boost vaccination Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Influenza A viruses (IAV) encode two important envelope glycoproteins that are targeted by the immune system of the host. The hemagglutinin (HA) is a trimeric transmembrane protein with a large ectodomain that is structured into a variable globular head domain and a more conserved stalk region. The globular head domain harbors the receptor-binding pocket that interacts with sialic acid residues on cellular glycoproteins and glycolipids 1 . Most virus-neutralizing antibodies that are produced by the immune system in response to IAV infection are directed to the globular head and interfere with the receptor-binding activity of HA 1,2 . However, IAV are highly mutable, and the immune response frequently selects for viral mutants that are characterized by amino acid substitutions in the globular head region, which prevents binding of neutralizing antibodies, a phenomenon known as “antigenic drift” 3 . Because of this antigenic drift, seasonal influenza vaccines need to be updated every year according to WHO recommendations which are published about six months before the upcoming influenza season. Sometimes this prediction is inaccurate and a mismatch between the vaccine strain and presently circulating seasonal IAV leads to low vaccine efficacy 4 , 5 . Thus, there is a need for vaccines that provide efficient protection against antigen-drifted IAV. Neuraminidase (NA) is the second important antigen of IAV. The tetrameric transmembrane protein exhibits sialidase activity which is essentially required for release of progeny virus from the infected cell membrane 6 , as well as for penetration of the mucus-rich layers covering the respiratory epithelia of the host 7 . Antibodies that inhibit NA sialidase activity can efficiently interfere with virus dissemination and thus have protective potential 8 – 10 . Not surprisingly, NA is also subject to antigenic drift 11 – 13 . Of note, the most used vaccines to control seasonal influenza epidemics are inactivated split IAV standardized for hemagglutinin (HA) content, whereas the protective features of the NA are usually neglected 14 . In our previous work, we developed a novel prime/boost vaccination strategy comprising of an intramuscular prime with recombinant RNA replicon particles composed of G protein-deleted vesicular stomatitis virus (VSVΔG) vector encoding the H1 antigen of the pandemic A/Hamburg/4/2009 (H1N1) strain (H1N1 HH4/09 ) followed by an intranasal boost using a live-attenuated influenza vaccine (LAIV) based on a modified H1N1 HH4/09 characterized by a truncated NS1 protein and lack of PA-X protein expression (NS1(1-126)-ΔPAX) 15 . This prime/boost vaccination protocol elicited a strong systemic and mucosal immune response in the porcine model and resulted in sterilizing immunity against challenge infection with the homotypic H1N1 HH4/09 strain. In addition, priming with H1-recombinant RNA replicon particles controls replication and shedding of LAIV, thereby adding to vaccine safety 16 . In the present study, we investigated the efficacy of the heterologous RNA replicon prime/LAIV boost strategy in protecting pigs against antigen-drifted H1N1 virus and compared it with a homologous RNA replicon prime/RNA replicon boost strategy. To achieve a broader immune response, a cocktail of two VSV-based RNA replicon particles was used for intramuscular priming of the animals, VSVΔG(H1) and VSVΔG(N1), which encode the HA and NA antigens of H1N1 HH4/09 , respectively. In the homologous prime/boost strategy, the animals were immunized a second time with the RNA replicon particles, while in the heterologous strategy, they were immunized intranasally with LAIV NS1(1-126)-ΔPAX. The immune response of the animals was examined for H1-specific virus-neutralizing antibodies as well as N1-specific antibodies showing sialidase-inhibitory activity. Finally, the animals were challenged with a reassortant virus encoding the HA and NA antigens of the antigen-drifted A/Victoria/2570/2019 (H1N1) (H1N1 Vic/19 ) strain. Our results suggest that while both prime/boost strategies efficiently controlled replication and nasal shedding of the antigen-drifted virus, the robust N1-specific response induced by the VSV replicons turned out to be broadly protective, also inhibiting zoonotic H5N1 virus isolated from infected dairy cows. Results Evaluation of homologous and heterologous prime/boost vaccination strategies in the pig animal model Specific pathogen-free (SPF) pigs (8 to 10 weeks of age) were randomly distributed into 4 groups with 5 animals each (Fig. 1 ). Animals of groups A and C were first immunized via the intramuscular (i.m.) route with VSV*ΔG, RNA replicon particles encoding the irrelevant GFP protein, while animals of groups C and D were immunized (i.m.) with VSVΔG(H1) and VSVΔG(N1) replicon particles (Fig. 1 ). Four weeks after the primary immunization, the pigs were boosted with either VSV*ΔG (group A) or VSVΔG(H1) and VSVΔG(N1) replicon particles (group B), while animals of groups C and D were boosted via the intranasal route with the LAIV NS1(1-126)-ΔPAX 15 , 16 . Vaccination of the animals with VSV replicon particles or LAIV was well tolerated by the animals and did not cause any significant clinical symptoms ( Supplementary Fig. 1, Supplementary Table 1 ). Nasal swab samples were collected on the following days from the animals of groups C and D to monitor shedding of the LAIV. In addition, three immunologically naïve pigs were added to each group C and group D two days after the booster vaccination to monitor potential transmission of the virus to the sentinel animals (Fig. 1 ). Four weeks after the second vaccination, the pigs were infected via the intranasal route with a reassortant virus containing the genomic segments 4 (HA) and 6 (NA) of A/Victoria/2570/2019 (H1N1) (H1N1 Vic/19 ) and the remaining 6 genomic segments of A/Hamburg/4/2009 (H1N1) (H1N1 HH4/09 ). The Victoria strain has been recommended as a component of the tetravalent influenza vaccine for use in the 2021 and 2022 southern hemisphere seasons 17 and for the 2022/2023 season in the northern hemisphere 18 . Compared to the original 2009 pandemic H1N1 virus, the HA and NA antigens of this 2019 H1N1 virus were characterized by several amino acid substitutions in their ectodomains ( Supplementary Figs. 2, 3 ). The second immunization boosts the neutralizing antibody response against antigen-drifted H1N1 virus To monitor H1N1-specific antibody responses, serum samples were prepared prior to vaccination (d0, pre-immune serum), 4 weeks after the primary vaccination (d28, prime serum) and 4 weeks after the second immunization (d56, boost serum) (Fig. 1 ), and analyzed for virus-neutralizing activity against H1N1 HH4/09 (Fig. 2 a) and the H1N1 HH4/09 /H1N1 Vic/19 6:2 reassortant virus encoding the segments 4 and 6 of H1N1 Vic/19 (Fig. 2 b). None of the SPF pigs had pre-existing neutralizing antibodies to either of the two viruses. Pigs of group A that had been immunized with the control vaccine VSV*ΔG did not develop any H1N1-neutralizing antibodies, as expected (Fig. 2 a, b). In contrast, animals of group B developed significantly high titers of H1N1 HH4/09 -neutralizing antibodies (geometric mean ND 50 = 485) after the first immunization with VSVΔG(H1) and VSVΔG(N1) replicon particles. The ND 50 titer increased further following the second immunization with the same vaccine (geom. mean ND 50 titer = 1358), however, the difference between the ND 50 titers of the prime and boost sera was not statistically significant (Fig. 2 a). When the immune sera of group B were tested for neutralizing activity against the H1N1 HH4/09 /H1N1 Vic/19 6:2 reassortant virus, the prime sera showed only very low neutralizing activity (geom. mean ND 50 = 6), which increased significantly after the second administration of the vaccine (geom. mean ND 50 = 277) (Fig. 2 b). When pigs were first immunized (i.m.) with VSV*ΔG and subsequently with the LAIV via the intranasal route (group C, d56), only low H1N1 HH4/09 -neutralizing serum antibody titers were detected (geom. mean ND 50 = 30) (Fig. 2 a), and inhibitory activity against the H1N1 HH4/09 /H1N1 Vic/19 6:2 reassortant virus was even lower (geom. mean ND 50 = 4) (Fig. 2 b). However, when the animals were first primed (i.m.) with VSVΔG(H1) and VSVΔG(N1) and then intranasally immunized with the LAIV (group D), geometric mean ND 50 values against the antigen-drifted H1N1 HH4/09 /H1N1 Vic/19 6:2 reassortant virus increased from 6 (d28, after prime) to 276 (d56, after boost) (Fig. 2 b), while neutralizing activity against the homotypic H1N1 HH4/09 strain was already high after the first immunization (geom. mean ND 50 = 365), and increased only marginally following immunization with LAIV (geom. mean ND 50 = 958) (Fig. 2 a). Finally, we tested whether the boost sera of group B would show some neutralizing activity against A/cattle/Texas/063224-24-1/2024 (H5N1) (H5N1 Tex/24 ), however, no significant inhibition of this virus was observed (Fig. 2 c). In conclusion, our findings suggest that a prime/boost vaccination regimen is important to produce robust levels of serum antibodies with neutralizing activity against antigen-drifted H1N1 virus. Vaccinated pigs produce N1-specific antibodies with sialidase-inhibitory activity To measure the functional antibody response triggered by the VSVΔG(N1) vaccine component, we performed an enzyme-linked lectin assay (ELLA) to detect antibodies inhibiting the enzyme activity of the N1 neuraminidase. The assay is based on immobilized fetuin as substrate and biotin-labeled peanut agglutinin (PNA) as probe for the desialylated product of the enzyme reaction 19 , 20 . Since the immune sera of immunized pigs also contain H1-specific antibodies that could sterically block the binding of N1-specific antibodies to the virion 10 , the test was performed with H5N1 HH4/09 , a bovine H5N1 virus with its genome segment 6 (NA) replaced by the corresponding segment of H1N1 HH4/09 . For biosafety reasons, the proteolytic cleavage site in the H5 hemagglutinin was converted into a monobasic motif as found in low-pathogenic H5Nx influenza viruses 21 . Pigs that had been vaccinated with the VSV*ΔG control vaccine (group A) did not develop antibodies with inhibitory activity against N1 HH4/09 (Fig. 3 a). Likewise, pigs immunized via the intranasal route with the LAIV (group C) did not develop significant levels of serum antibodies with N1 inhibitory activity. In contrast, pigs developed geometric mean sialidase inhibitory antibody titers of IC 50 = 1267 (group B) and IC 50 = 1283 (group D) after the prime immunization (i.m.) with the VSVΔG(H1) plus VSVΔG(N1) vaccine. The second intramuscular administration of the vaccine significantly boosted the N1-specific antibody response in group B (geom. mean IC 50 = 7098), while the intranasal boost with the LAIV (group D) did not result in significantly increased inhibitory antibody titers (geom. mean IC 50 = 2065). To analyze the inhibitory activity of the immune sera against N1 sialidases from other influenza viruses, we also generated H5N1 reassortant viruses encoding the N1 sialidases of either H5N1 Vic/19 or H5N1 Tex/24 and the N2 sialidase of A/chicken/Ibaraki/1/2005 (H5N2) (H5N2 Iba/05) . Interestingly, the immune sera from group B pigs showed similarly high inhibitory activity against all three N1 sialidases but did not inhibit the N2 sialidase (Fig. 3 b). These results show that the intramuscular immunization with the VSVΔG(N1) vaccine component led to the production of N1-specific serum antibodies that have potent sialidase inhibitory activity, even against distantly related N1 such as the one from the bovine H5N1 Tex/24 virus. Antibodies to the N1 neuraminidase inhibit the release of H5N1 from infected MDCK cells Immune sera from vaccinated pigs of group B did not show neutralizing activity against H5N1 Tex/24 (see Fig. 2 c) but showed potent inhibition of N1 Tex/24 sialidase activity (see Fig. 3 b). To study the impact of N1 sialidase inhibition on the release of infectious H5N1 Tex/24 , we infected 96-well MDCK cell cultures with 100 TCID 50 /well of H5N1 Tex/24 and then added cell culture medium containing serially diluted immune sera. These serum pools were prepared for all four vaccine groups four weeks after the second immunization. At 24 and 48 hours p.i., the infectious titers of H5N1 Tex/24 released into the cell culture supernatants were determined on MDCK cells by end point titration. Immune sera of groups A and C did not inhibit the release of H5N1 Tex/24 from infected MDCK cells, independently of the serum concentration used (Fig. 4 a, b). In contrast, immune sera from group B showed a concentration-dependent inhibition of H5N1 Tex/24 release. Using the immune serum at a dilution of 1:20, a remarkable reduction of infectious titer by 6 log 10 was achieved at 24 h (Fig. 4 a), which was still present at 48 h p.i. (Fig. 4 b). Likewise, pooled group D immune serum mediated concentration-dependent inhibition of H5N1 Tex/24 release, however, with general lower efficacy compared to group B immune serum. Analysis of the sera from individual animals allowed us to determine the serum concentration leading to 90% inhibition of H5N1 Tex/24 release at 24 hours p.i. ( Fig. 4 c ). The geometric mean IC 90 titer was 896 for group B and 160 for group D, whereas group A and C sera had no significant impact on virus release. Release of a HPAI H5N4 virus was not inhibited by the group B serum pool, indicating that the inhibitory activity of the immune sera is NA subtype specific. At 48 h p.i., when the cell culture supernatant had been removed, the cells were fixed with formalin and infected cells visualized by indirect immunofluorescence using a monoclonal antibody directed to the influenza NP antigen. In MDCK cells incubated with group A or group C serum, H5N1 Tex/24 infection spread throughout the whole monolayer, whereas in cells incubated with group B or D serum the infection remained restricted to distinct foci (Fig. 4 d). Collectively, these results demonstrate that the VSVΔG(N1) vaccine component induces N1-specific serum antibodies with potent inhibitory activity against H5N1 Tex/24 . Intramuscular priming of pigs eliminates LAIV shedding In a recent study we showed that the prolonged shedding of the LAIV NS1(1-126)-ΔPAX from the upper respiratory tract of vaccinated pigs can be reduced if the animals were first primed with VSVΔG(H1) replicon particles 22 . To confirm this finding and to see whether the vaccination of the pigs with VSVΔG(H1) in combination with VSVΔG(N1) could completely eliminate LAIV shedding, the vaccinated pigs of groups C and D were monitored for a total of 16 days by analyzing nasal swab samples by RT-qPCR for the presence of viral RNA (Fig. 5 a) and infectious virus (Fig. 5 b). Using these methods, we detected significant LAIV shedding by animals of group C, which lasted for several days. The three non-vaccinated pigs that were kept for three weeks with the animals of group C seroconverted to the influenza NP antigen (Fig. 5 c), indicating that LAIV was transmitted to these sentinels. In contrast, if the pigs were first primed with the VSVΔG(H1)/VSVΔG(N1) vaccine, only some viral RNA was detected in nasal swabs at day 1 and 2 after nasal administration of the LAIV (Fig. 5 a), and no shedding of infectious virus (Fig. 5 b) or transmission to sentinels was detected (Fig. 5 c). These data confirmed that intramuscular priming with the H1/N1 RNA replicon particles efficiently controlled replication of the LAIV in the upper respiratory tract. Vaccination of pigs efficiently prevents replication of antigen-drifted H1N1 virus in the respiratory tract Four weeks after the second vaccination, the animals were challenged via the intranasal route with 10 6 infectious units/pig of the H1N1 HH4/09 /H1N1 Vic/19 6:2 reassortant virus. None of the animals, including those in control group A, showed an elevated rectal temperature ( Supplementary Fig. 1 ) or other clinical symptoms ( Supplementary Table 2 ) as a result of the infection. To monitor the shedding of challenge virus, nasal swab samples were collected daily for a total of 5 days post infection and analyzed for the presence of viral RNA (Fig. 6 a) and infectious virus (Fig. 6 b). As expected, all animals of control group A displayed shedding of challenge virus starting from day 2 and lasting until the end of the experiment at day 5 post infection (Fig. 6 a, b). In contrast, no viral RNA was detected in swab samples taken from group C animals at any of the post challenge days, and only some swab samples collected from pigs of groups B and D were positive for viral RNA on some days (Fig. 6 a). However, no infectious virus was detected in the nasal swabs collected from the pigs from groups B, C, and D (Fig. 2 b). In line with these findings, viral RNA was detected in tonsils, mesenteric and tracheobronchial lymph nodes and lung lobes in most animals of group A but was essentially absent from the corresponding tissues of animals of group B, C, and D ( Fig. 6 c ). These results show that both the heterologous and homologous prime/boost vaccination regimens are effective in preventing the replication and shedding of antigen-drifted virus. Discussion Heterologous prime/boost immunization Antigen-drift represents a major hurdle for the efficacy of conventional influenza vaccines that rely on inactivated split viruses and are standardized to the amount of purified HA antigen. In the present work, we show that intranasal immunization of pigs (group C) with the LAIV NS1(1-126)-ΔPAX triggers an immune response that efficiently controlled the replication of a reassortant virus bearing the HA and NA envelope glycoproteins of the antigen-drifted H1N1 Vic/19 strain. Although intranasal immunization with LAIV induced relatively low levels of virus-neutralizing serum antibodies, previous work with the same LAIV showed that intranasal immunization of pigs with this vaccine induces local immune responses including secretion of H1N1-specific IgA into the mucosal tissues and cellular immune responses that are expected to contribute to its potent inhibition of challenge virus replication in the mucosa 22 . However, the prolonged shedding of LAIV via the respiratory tract and transmission to sentinel pigs was identified as a safety issue 22 – 24 . This problem was solved by first immunizing the animals via the intramuscular route with VSV-based RNA replicon particles encoding HA 22 . This immune priming induced high levels of systemic immunity that efficiently controlled LAIV shedding without preventing the LAIV from boosting neutralizing antibodies. In the present study, immune priming with both H1 and N1 antigen also eliminated LAIV shedding. Still, the boost vaccination using the LAIV significantly enhanced the systemic immune response against the antigen-drifted HA Vic/19 protein. Furthermore, the inclusion of VSV replicons encoding the N1 HH4/09 antigen resulted in the induction antibodies that inhibited the sialidase activity of N1 HH4/09 and N1 Vic/19 equally well. As a result, this vaccination protocol induced robust cross-protective immunity, even preventing shedding of heterologous challenge virus from the respiratory tract. Homologous prime/boost immunization The first immunization (i.m.) with the combined VSVΔG(H1) and VSVΔG(N1) vaccine resulted in high antibody titers with neutralizing activity against homologous H1N1 HH4/09, which increased only moderately after administration of the second vaccine dose. In contrast, the neutralizing activity against antigenically drifted H1N1 Vic/19 strain was strongly boosted by the second dose. This broadened immunity was likely driven by memory B cells undergoing affinity maturation upon antigen re-exposure 25 , 26 . Notably, re-stimulating memory B cells with the same antigens enhances not only the strength and quality but also the longevity of antibody responses 27 . Another important finding of our study was that homologous prime/boost strategy using the combined replicon particle vaccines eliminated the shedding of the antigen-drifted H1N1 challenge virus, even though the vaccines were administered via the intramuscular route. 28 . Possibly, H1- and N1-specific serum IgG was secreted into the mucosal tissues of the respiratory tract via the neonatal Fc receptor (FcRn) which is expressed in mammalian species also at adult life 29 . In mice, passive intravenous transfer of anti-influenza immunoglobulins indicated that high serum IgG levels can efficiently reduce nasal influenza virus shedding 28 , suggesting that a fraction of the serum IgG can be secreted to mucosal surfaces. Thus, the strong immune response induced by intramuscular immunization with the RNA replicon particles is the likely key factor preventing challenge virus replicating in the respiratory mucosa and, consequently, virus shedding. Importance of NA-specific antibodies in protection In contrast to our previous study in which the VSVΔG(H1) vaccine was used for immunization of pigs 22 , the present work made also use of VSVΔG(N1) replicon particles encoding N1 HH4/09 . The N1-specific antibodies elicited by this vaccine potently inhibited the sialidase activity of the homologous N1 HH4/09 but also the sialidase activity of N1 Vic/19 and N1 Tex/24 , although the latter shows only 88% amino acid identity with N1 HH4/09 ( Supplementary Fig. 2 ). This suggests that the N1-specific antibodies bound to epitopes that are conserved in this antigen. There is evidence that the antigenic drift associated with NA is slower than that of the HA antigen, possibly because of functional constraints, i.e. only mutations that do not significantly impair sialidase activity are tolerated 12 , 30 . NA sialidase activity is crucial for the efficient release of progeny influenza virus from the infected host cell 31 , 32 but also helps the virus to penetrate the mucus layers covering the epithelial cells of the respiratory tract 33 , 34 . Thus, the antibodies induced by VSVΔG(N1) likely contributed to immune protection of the pigs by inhibiting virus release and spread. Indeed, previous work has shown that VSVΔG(NA) replicon particles are protective even if used as a stand-alone vaccine 10 , 35 . The observation that immune sera of pigs immunized with the combined VSVΔG(H1) and VSVΔG(N1) replicon particles potently inhibited release and spread of H5N1 Tex/24 , despite lacking virus-neutralizing activity, underscores the enhanced broad cross-reactivity achieved by targeting NA in vaccination. In recent years, H5N1 HPAI viruses of the clade 2.3.4.4b have spread worldwide and led to the death of millions of birds of numerous species 36 . In addition, clade 2.3.4.4b viruses showed the remarkable potential to infect various mammalian species 37 , 38 , including humans 39 . While infection of cats by H5N1 caused neurological symptoms with often fatal outcome 40 , infection of humans was mostly associated with mild symptoms such as conjunctivitis 41 . There is evidence that infection with human influenza viruses elicits broadly cross-reactive NA-specific antibodies that protect from infection by HxN1 viruses in vitro and in vivo 42 , 43 . The protective effect of prior infection with 2009 pandemic H1N1 virus on the pathogenesis and transmission of clade 2.3.4.4b H5N1 virus was also observed in the ferret model 44 , 45 . These findings imply that children who have not experienced infection with seasonal H1N1 virus yet might be at higher risk to develop severe symptoms if infected with H5N1 HPAI. Indeed, severe disease following infection with clade 2.3.4.4b genotype D1.1 was documented for a 13-year-old teenager in 2024 in Canada 46 and a fatal infection of a 3-year-old girl was reported in 2025 in Mexico 47 . However, it is not known whether these children were more susceptible to H5N1 infection because they had no pre-existing immunity to influenza viruses in general. Taken together, the present study not only confirms the efficacy and safety of influenza vaccines based on non-replicating RNA replicon particles in the porcine animal model 22 , 48 , 49 , but demonstrates that the high levels of neutralizing anti-HA as well as the NA-targeting antibodies ensure broad systemic and mucosal protection against antigen-drifted influenza viruses. These properties represent a significant improvement compared to currently used inactivated seasonal influenza vaccines 50 , 51 . Methods Cells Madin-Darby canine kidney type II cells (MDCK-II) were kindly provided by Georg Herrler (University of Veterinary Medicine, Hannover, Germany) and maintained with minimum essential medium (MEM, Thermo Fisher Scientific, Basel, Switzerland; cat. no. 31095-029) supplemented with 5% of fetal bovine serum (FBS; Pan Biotech, Aidenbach, Germany; cat. no. P30-3033). Human embryonic kidney (HEK) 293T cells (American Type Culture Collection (ATCC), Manassas, USA; cat. no. CRL-3216) were maintained with Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific; cat. no. 32430-027) supplemented with 10% FBS. Baby hamster kidney 21 (BHK-21) fibroblasts were obtained from ATCC (cat. no. CCL-10) and maintained in Glasgow’s minimal essential medium (GMEM, Thermo Fisher Scientific, cat. no. 21710-025) supplemented with 5% FBS. BHK-G43 cells, a transgenic BHK-21 cell clone expressing VSV glycoprotein G in a regulated manner 52 , was maintained in GMEM supplemented with 5% FBS. All cells were grown at 37°C in a 5% CO 2 atmosphere. Viruses The HPAI virus A/cattle/Texas/063224-24-1/2024 (H5N1) (H5N1 Tex/24 ), clade 2.3.4.4b, genotype B3.13 (GISAID accession number: EPI_ISL_19155861), was kindly provided by Diego Diel (Cornell University, Ithaca, NY, USA). This virus was originally isolated from the milk of infected dairy cows in Texas, USA 53 . A/Buzzard/Switzerland/V0052/2021 (H5N4) (H5N4 Swi/21 ) was isolated by IVI Mittelhäusern from a brain biopsy sample taken from a common buzzard that was found dead close to Lake Constance in 2021. The 2009 pandemic virus strain A/Hamburg/4/2009 (H1N1) (H1N1 HH4/09 ) was produced by reverse genetics using the 8-plasmid system 15 . The live-attenuated influenza vaccine (LAIV) NS1(1-126)-ΔPAX is based on a modified H1N1 HH4/09 and encodes a truncated NS1 protein and is unable to drive expression of the accessory PA-X protein 15 . VSV*ΔG is a propagation-defective vesicular stomatitis virus in which the glycoprotein (G) gene has been replaced by the green fluorescent protein (GFP) gene 54 . Generation of recombinant influenza viruses The cDNAs encoding the genomic segments 4 and 6 of A/Victoria/2570/2019 (H1N1) (GenBank acc. nos. WEY08940 and WEY08939, respectively) and the cDNAs of the eight genomic segments of A/bovine/Texas/24-029328-02/2024 (H5N1) (GenBank accession nos. PP599470–PP599477) 55 were synthesized by Genscript Biotech (Piscataway, New Jersey, USA). The synthetic cDNA of RNA segment 4 encoded an HA containing the monobasic cleavage site P 337 LRETR↓GLF rather than the polybasic cleavage site P 337 LREKRRKR↓GLF found in the authentic virus. All cDNAs were amplified with Phusion DNA and cloned into the Bsm BI restriction site of the pHW2000 plasmid 54 using the In-Fusion cloning system (Takara Bio, Saint-Germain-en-Laye, France, cat. no. 638948). The primary nucleotide sequence of each of the cloned segments was verified by Sanger sequencing. Infectious viruses were generated by transfection of HEK 293T/MDCK cell co-cultures using the respective eight genomic cDNAs cloned into the pHW2000 vector and Lipofectamine 2000 (Life Technologies, cat. no. 11668019) as transfection reagent. In this way, the following recombinant viruses were generated: A virus containing segments 4 and 6 of H1N1 Vic/19 and the remaining six segments of H1N1 HH4/09 , a H5N1 Tex/24 with a monobasic proteolytic cleavage site in HA, and three reassortant viruses encoding segment 6 of either H1N1 HH4/09 , H1N1 Vic/19 , or H5N1 Tex/24 and the remaining genomic RNA segments of (H5 mb N1) Tex/24 . The rescued viruses were passaged two times on MDCK cells with FBS-deficient medium containing 1% penicillin/streptomycin and 1 µg/mL of acetylated trypsin (Merck KGaA, Darmstadt, Germany, cat. no. 4370285). Virus stocks were stored in aliquots at -70°C in the presence of 5% FBS. Titration of influenza A viruses For titration of IAV that had a monobasic cleavage site in their HA, MDCK cells grown in 96-well tissue culture plates were inoculated in duplicate with 40 µL per well of serial 10-fold virus dilutions for 1 h at 37°C. Thereafter, 160 µL of MEM containing 1% methylcellulose were added to each well, and the cells incubated for 24 h at 37°C. The cells were fixed for 30 min with 4% formalin in PBS, permeabilized with 0.25% (v/v) of Triton X-100, and incubated for 60 min with a monoclonal antibody directed to the influenza nucleoprotein (NP) antigen (American Type Culture Collection, Manassas, Virginia, USA, ATCC HB-65, clone H16-L10-4R5, diluted 1:50 in PBS), and subsequently for 60 min with goat anti-mouse IgG conjugated with Alexa Fluor-488 (Life Technologies, Zug, Switzerland; diluted 1:500 in PBS). Infected cells were detected by fluorescence microscopy (Observer Z1 microscope, Zeiss, Feldbach, Switzerland), and infectious virus titers were calculated and expressed as focus-forming units per milliliter (ffu/mL). For titration of the HPAI H5N1 Tex/24 , MDCK cell monolayers in 96-well tissue culture plates were incubated in quadruplicates with serially diluted virus (100 µL/well) for 48 h at 37°C. The cells were washed once with PBS (200 µL/well) and fixed for 1 h at 21°C with 4% of buffered formalin containing 0.1% (w/v) crystal violet (Merck KGaA, cat. no. 1.01408). The plates were washed with tap water and dried. Virus titer was calculated using the Spearman-Kärber method and expressed as tissue culture infectious dose 50% (TCID 50 ) 56 . Generation of recombinant VSV vector vaccine For generation of the VSVΔG(H1) and VSVΔG(N1) vaccine components, the HA and NA genes of H1N1 HH4/09 (GenBank acc. nos. GQ166213 and GQ166217, respectively) were amplified by PCR using the Phusion DNA polymerase and inserted into the Mlu I and Nhe I sites of the pVSV*ΔG(HA H5−HP ) plasmid 57 , resulting in the pVSVΔG(H1) and pVSVΔG(N1) plasmids, respectively. VSVΔG(H1) and VSVΔG(N1) replicon particles were generated from transfected cDNA 49 , 58 , 59 and propagated on BHK-G43 cells expressing the VSV G protein in a regulated manner 52 . Virus stocks were stored in aliquots at -70°C. Infectious virus titers were determined on BHK-21 cells by end point dilution. Infected cells were detected 20 h p.i. by indirect immunofluorescence using a monoclonal antibody (mAb 23H12) directed to the VSV matrix protein (KeraFast, Boston, MA, cat. no. EB0011) 60 . For production of vaccine batches, BHK-G43 cells were seeded into five T150 flasks and maintained in 40 mL/flask of GMEM medium with 5% FBS. When confluency was reached, the medium was aspirated and the cells maintained at 37°C for 6 h with 40 mL/flask of GMEM containing 10 − 9 M of mifepristone. VSVΔG(H5 mb ) was added at a multiplicity of infection (moi) of 0.05 ffu/cell and incubated with the cells for 20 h at 37°C. The cell culture supernatant was transferred to 50-mL Falcon tubes and cell debris removed by centrifugation (1000 × g, 15 min, 4°C). The replicon particles were pelleted from the cleared supernatant by ultracentrifugation (105`000 × g, 60 min, 4°C) and resuspended in 20 mL of PBS. The VSVΔG(H1) and VSVΔG(N1) replicon particles were stored in aliquots at -70°C. Animal experiments The animal experiments were performed according to Swiss laws (the Animal Welfare Act TSchG SR 455, the Animal Welfare Ordinance TSchV SR 455.1, and the Animal Experimentation Ordinance TVV SR 455.163). All experiments were reviewed by the committee on animal experiments of the canton of Bern and were approved by the cantonal veterinary authority under license number BE15/2024 . Twenty 10-week-old specific pathogen-free (SPF) Swiss Large White pigs from the breeding facility of the Institute of Virology and Immunology IVI (Mittelhäusern, Switzerland) were randomly divided into four groups (A – D), each containing five pigs of both sexes (n = 5). After an acclimatization period of five days, the animals of groups A and C received 2 mL of VSV*ΔG suspension (4 × 10 8 ffu/mL) that were injected into the m. brachiocephalicus (neck muscle) (Fig. 1 ). The animals of groups B and D were immunized by injection of 2 mL (4 × 10 8 ffu/mL) of VSVΔG(H1) and 2 mL (4 × 10 8 ffu/mL) of VSVΔG(N1) suspension into different sites of the neck muscle. Body temperature and clinical symptoms were monitored daily for seven days, and serum samples were prepared at day 28 and day 56 after primary immunization (Fig. 1 ). At day 28, the animals of group A received a second dose (2 mL, 4 × 10 8 ffu/mL) of the control vector VSV*ΔG while animals of group B were immunized (i.m.) with VSVΔG(H1) and VSVΔG(N1) replicon particles using the same doses as for the first time. Animals of groups C and D were immunized via the nasal route with 5 mL of PBS containing 2 × 10 4 ffu/mL of LAIV. To this end, an intranasal mucosal atomization device (MAD Nasal, Teleflex Medical Europe Ltd., Ireland, cat. no, MAD300) plugged to a 10-mL syringe was employed. After the first and the second immunization, the rectal temperature of the pigs and potential vaccine-mediated adverse effects were recorded daily for a total of 10 days. Blood (10 mL) was collected prior to vaccination (day 0), 28 days after prime vaccination, and 28 days after the booster immunization (day 56). Serum was prepared after coagulation of the blood and stored at -20°C prior to analysis by virus neutralization, sialidase inhibition and virus release inhibition tests. To monitor shedding of LAIV, two nasal swabs were taken from each animal of groups C and D prior to vaccination (day 28) and at days 29, 30, 32, 34, 36, 38, 40, 42, 44. One swab sample was soaked in 0.75 mL of MEM supplemented with penicillin/streptomycin (Life Technologies, cat. no. 15140-122) and amphotericin B (Life Technologies, cat. no. 15290018), while the second swab was soaked in 0.75 mL of RA1 lysis buffer (Macherey-Nagel, Düren, Germany; cat. no. 740961) containing 1% (v/v) β-mercaptoethanol. Swab-soaked media and lysates were stored at -70°C prior to analysis by virus titration and RT-qPCR, respectively. To monitor transmission of the LAIV, three sentinel animals were co-housed with each group C and group D at day 30 of the experiment (48 h after the second immunization). The sentinel animals were euthanized at day 54 and their serum tested for the presence of NP-specific antibodies by ELISA. On day 56, animals in groups A to D were infected intranasally with 2 ml PBS containing 10 6 ffu/ml of the H1N1 HH4/09 /H1N1 Vic/19 6:2 reassortant. After infection, rectal temperature and clinical symptoms, including liveliness, body tension, body shape, appetite, nasal discharge, respiratory rate, expiratory effort, wheezing/coughing and diarrhea, were recorded daily for a total of 5 days. A clinical scoring system was used to assess disease severity and determine the humane endpoint: 0 = no symptoms, 1 = mild symptoms, 2 = moderate symptoms, 3 = severe symptoms. The animals were euthanized immediately if they reached a total clinical score of 9, or if a clinical score of 3 was reached for one or more of the clinical parameters. In addition, nasal swab samples were taken daily and tested for the presence of viral RNA using RT-qPCR. Five days after infection, the animals were killed by electrocution and subsequent exsanguination. Immediately after exsanguination, biopsy material was taken from the tonsils, mesenteric lymph nodes, tracheobronchial lymph nodes and the right cranial lobe of the lung and tested for the presence of viral RNA using RT-qPCR. Sialidase inhibition assay The heat-inactivated immune sera of all animals in a group were mixed to obtain group-specific serum pools for both the primary and the booster vaccinations. Starting with a dilution of 1:20, four-fold serially diluted serum pools were prepared in 96-well plates using PBS as diluent. To 50 µL of each serum dilution, 50 µL of NA reassortant H5N1 virus (10 7 ffu/mL) were added (in duplicates) and incubated for 30 min at 37°C, The serum-virus mixture (100 µL) was then transferred to Nunc MaxiSorp 96-well plates (Thermo Fisher Scientific) that had been coated for 24 h at 4°C with 10 µg/well of bovine fetuin (Merck KGaA, Darmstadt, Germany, cat. no. F2379) and subsequently blocked with 1% bovine serum albumin (BSA, Fluka Chemie GmbH, Buchs, Switzerland, cat. no. 05480). The microtiter plates with the immobilized fetuin were incubated with the serum-virus mixtures for 20 h at 37°C and then washed three times with 250 µL/well of PBS containing 0.05% Tween 20 (PBS-T). Subsequently, 50 µL/well of PBS containing 0.1% BSA and 0.5 µg/mL of biotinylated peanut agglutinin (PNA, Merck KGaA cat. no. L6135) were added and incubated for 1 h at 21°C. The microtiter plates were washed three times with PBS-T before 50 µL/well of streptavidin-peroxidase conjugate (Dako, Glostrup, Denmark; 1:5000 in PBS) were added and incubated with the plates for 30 min at 21°C. The wells were washed as above again and 50 µL of 3,3′,5,5′-Tetramethylbenzidine (TMB) peroxidase substrate (Merck KGaA, cat. no. T4444) was added to each well. The reaction was stopped by adding 50 µL/well of 1 M hydrochloric acid (Merck KGaA, cat. no. 1.09057.1000) and absorption was measured at 450 nm using the GloMax® Discover microplate reader (Promega Corporation, Madison, WI, USA). The OD 450 values were plotted against the serum dilution factors, and the dilution factor resulting in 50% inhibition (inhibitory concentration 50% [IC 50 ]) was calculated by non-linear regression analysis (curve fitting). Virus neutralization test Porcine sera were heated for 30 min at 56°C to inactivate complement factors. Serial two-fold dilutions in MEM medium were added in quadruplicates to 96-well microtiter plates (50 µL/well). To each well, 50 µL of H1N1 HH4/09 , H1N1 Vic/19 or H5N1 Tex/24 (2000 ffu/mL) were added and incubated for 1 h at 37°C. The antibody/virus mix was incubated for 1 h at 37°C and 5% CO 2 with MDCK-II cells that were grown to confluence in 96-well cell culture plates. Thereafter, the cells were washed once and incubated for 24 h at 37°C with 100 µL/well of MEM medium. The cells were fixed with 4% formalin and permeabilized with PBS containing 0.25% Triton X-100. Virus-infected cells were visualized by indirect immunofluorescence using a monoclonal antibody directed to the influenza nucleoprotein (NP) antigen (American Type Culture Collection, Manassas, Virginia, USA, ATCC HB-65, clone H16-L10-4R5, diluted 1:50 with PBS) and goat anti-mouse IgG conjugated with Alexa Fluor-488 (Life Technologies, Zug, Switzerland; diluted 1:500 in PBS) 61 . The 50% neutralizing dose (ND 50 ) was calculated using the Spearman-Kärber method 56 . Virus release inhibition assay MDCK cells were grown in 96-well tissue culture plates and inoculated for 30 min at 37°C with 50 µl MEM containing 100 ffu of H5N1 Tex/24 . The cells were washed once with 200 µL of Dulbecco`s phosphate-buffered saline (DPBS; Life Technologies, cat. no. 14040-091) and incubated at 37°C with 200 uL of MEM cell culture medium containing serially two-fold diluted immune sera (boost) and supplemented with penicillin and streptomycin (Life Technologies, cat. no. 15140-122). At 24 h and 48 h post infection, 100 µL aliquots of cell culture supernatant were collected and frozen at -70°C. The number of infectious virus particles released was determined as described above (see section “Titration of influenza A viruses”). RT-qPCR Swab samples were directly transferred to 700 µl of RA1 lysis buffer (Macherey-Nagel, Düren, Germany, cat. no. 740961) containing 1% β-mercaptoethanol. Organs were homogenized in RA1 lysis buffer using a tissue bullet blender (Next Advanced Inc., Troy, NY, USA). Total RNA was extracted from the lysates using the NucleoMag Vet kit (Macherey-Nagel, cat.no. 744200) according to the manufacturer’s protocol. Reverse transcription from RNA to cDNA and real-time quantitative PCR (qPCR) were performed on a QuantStudio 5 real-time PCR system (Thermo Fisher Scientific) using the AgPath-ID One-Step RT-PCR kit (Life Technologies, Zug, Switzerland, cat. no. AM1005) with vRNA segment 7-specific oligonucleotide primers and probe 62 , 63 . Data were acquired and analyzed using the Design and Analysis Software v1.5.2 (Thermo Fisher Scientific). Statistical analysis Statistical analyses were performed using GraphPad Prism 10, version 10.1.2 (GraphPad Software, Boston, Massachusetts, USA). Unless noted otherwise, data are presented as scatter dot plots with geometric mean values and 95% confidence interval (CI) also indicated. Specific statistical tests such as the one-way or two-way ANOVA test were used to assess significant differences in serum antibody responses in vaccinated animals as indicated in the figure legends. P values < 0.05 were considered significant. Declarations Acknowledgements We like to thank the Swiss National Research Foundation (SNSF) and the European Union’s Horizon Europe Project 101136346 EUPAHW for financial support of this project. We are grateful to Daniel Brechbühl and Katarzyna Sliz for their support in animal experimentation. We like to thank Martin Schwemmle (University of Freiburg, Germany) and Yoshihiro Sakoda (University of Sapporo, Japan) for providing plasmids and Diego Diel (Cornell University, USA) for providing the bovine H5N1 virus isolate. Author contributions AS and GZ conceived the study and were responsible for funding acquisition; NR, OG and GZ conceptualized and conducted the animal experiments; RA, LB, and GZ generated recombinant influenza viruses and VSV replicon vaccines; LB performed RT-qPCR analysis, virus neutralization tests, virus release inhibition tests, and sialidase inhibition tests; LB and GZ analyzed and evaluated the data. GZ wrote the manuscript draft. All authors read and approved the final manuscript. Data availability All data generated or analyzed during this study are included in this published article and its supplementary information files. Raw data are available at Zenodo ( https://doi.org/10.5281/zenodo.17302034 ). Competing interests RA, GZ, and AS filed a patent application related to the intramuscular prime/intranasal boost vaccine described in this work. All other authors declare no competing interests. Funding This work has received funding from the Swiss National Research Foundation (SNSF), grant no. 189903 (A.S., G.Z.; https://data.snf.ch/grants/grant/189903) and was co-funded by the European Union’s Horizon Europe Project 101136346 EUPAHW by a grant to A.S.. The funders had no role in study design, data collection and analysis, decisions to publish or preparation of the manuscript. References Wu, N. C. & Wilson, I. A. A Perspective on the Structural and Functional Constraints for Immune Evasion: Insights from Influenza Virus. J Mol Biol 429 , 2694-2709, doi:10.1016/j.jmb.2017.06.015 (2017). Wu, N. C. & Wilson, I. A. Influenza Hemagglutinin Structures and Antibody Recognition. Cold Spring Harb Perspect Med 10 , doi:10.1101/cshperspect.a038778 (2020). Kim, H., Webster, R. G. & Webby, R. J. Influenza Virus: Dealing with a Drifting and Shifting Pathogen. Viral Immunol 31 , 174-183, doi:10.1089/vim.2017.0141 (2018). Morimoto, N. & Takeishi, K. 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A recombinant vesicular stomatitis virus replicon vaccine protects chickens from highly pathogenic avian influenza virus (H7N1). Vaccine 27 , 1174-1183, doi:10.1016/j.vaccine.2008.12.019 (2009). Thompson, D., Cismaru, C. V., Rougier, J. S., Schwemmle, M. & Zimmer, G. The M2 proteins of bat influenza A viruses reveal atypical features compared to conventional M2 proteins. J Virol 97 , e0038823, doi:10.1128/jvi.00388-23 (2023). Lenz-Ajuh, N. et al. Impact of pH and temperature in dairy processing on the infectivity of H5N1 avian influenza viruses. Int J Food Microbiol 441 , 111328, doi:10.1016/j.ijfoodmicro.2025.111328 (2025). Hofmann, M. A., Renzullo, S. & Baumer, A. Phylogenetic characterization of H5N1 highly pathogenic avian influenza viruses isolated in Switzerland in 2006. Virus Genes 37 , 407-413, doi:10.1007/s11262-008-0285-2 (2008). Spackman, E. et al. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J Clin Microbiol 40 , 3256-3260, doi:10.1128/JCM.40.9.3256-3260.2002 (2002). Additional Declarations Competing interest reported. RA, GZ, and AS filed a patent application related to the intramuscular prime/intranasal boost vaccine described in this work. All other authors declare no competing interests. Supplementary Files SupplementaryFig1.png Supplementary Fig. 1 | Recording of the rectal temperature in SPF pigs following vaccination and challenge infection. a-d The rectal temperature was recorded in all animals of the indicated groups (n = 5 animals/group) at the indicated day of the experiment. Mean values with SD (dotted lines) are shown. aRecording of rectal temperature following prime vaccination of groups A - D. bRecording of the rectal temperature in animals of groups A and B following boost vaccination. C Recording of rectal temperature in animals of group C and D (n = 5/group) after receiving the LAIV and in the sentinal pigs ( n = 3) that were co-housed with each of the groups. SupplementaryFig2.png Supplementary Fig. 2 | Alignment of HA primary amino acid sequences. The HA sequences of A/Hamburg/4/2009 (H1N1) (GenBank accession no. ACR10223) and A/Victoria/2570/2019 (H1N1) (GenBank accession no. WEY08940) are shown. The predicted signal sequences (amino acids 1-17) and transmembrane domains (amino acids 537-560) are depicted with a grey background. Amino acid positions differing between the two HA antigens are shown in bold letters. The amino acid identity of the two HA antigens is 95.5%. SupplementaryFig3.png Supplementary Fig. 3 | Alignment of NA primary amino acids sequences. Alignment of the NA amino acid sequences of A/Hamburg/4/2009 (H1N1) (GenBank accession no. ACR10227), A/Victoria/2570/2019 (H1N1) (GenBank accession no. WEY08939), and A/cattle/Texas/063224-24-1/2024 (H5N1) (GISAID acc. no. EPI_ISL_19155861) are shown. The predicted transmembrane domains (amino acids 7-30) are depicted with a grey background. Amino acid positions differing between the two NA antigens are shown in bold letters. SupplementaryTables.docx Cite Share Download PDF Status: Published Journal Publication published 28 Mar, 2026 Read the published version in npj Vaccines → Version 1 posted Editorial decision: Revision requested 28 Jan, 2026 Reviews received at journal 26 Jan, 2026 Reviewers agreed at journal 12 Jan, 2026 Reviews received at journal 19 Dec, 2025 Reviewers agreed at journal 02 Dec, 2025 Reviewers agreed at journal 28 Oct, 2025 Reviewers invited by journal 27 Oct, 2025 Editor assigned by journal 27 Oct, 2025 Submission checks completed at journal 23 Oct, 2025 First submitted to journal 09 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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SPF pigs (8-weeks-old) of mixed sex were randomly assigned to the indicated vaccine groups (n = 5 pigs/group) and were first immunized (i.m.) with either VSV*DG (groups A and C) or with VSVDG(H1) and VSVDG(N1) replicon particles (groups B and D) using the indicated doses. Four weeks after the primary immunization, the animals were boosted with either the virus-vectored H1 and N1 antigens (i.m.) or the LAIV (intranasal). Sentinel pigs (n = 3) were co-housed on day 30 with groups C and D to monitor transmission of LAIV. Four weeks after the booster vaccination, all animals of groups A to D were challenged intranasally with the H1N1\u003csub\u003eHH4/09\u003c/sub\u003e/H1N1\u003csub\u003eVic/19\u003c/sub\u003e 6:2 reassortant virus. Five days post challenge, all animals were euthanized, and lung biopsy samples were taken to determine viral RNA load by RT-qPCR. At the indicated times, blood samples were collected and sera prepared which were analyzed for virus-neutralizing and sialidase-inhibitory activity. In addition, oropharyngeal swab samples were collected (red arrows) and analyzed for the presence of infectious virus and viral RNA load. The piglet image was created in BioRender. Zimmer, G. (2025) https://BioRender.com/ai29p9b.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7818522/v1/1a96bf486575ade01a724a94.png"},{"id":95524900,"identity":"c75bb79d-d87f-4b96-96fd-a7518a08b3f9","added_by":"auto","created_at":"2025-11-10 10:03:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":31162,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetermination of virus-neutralizing serum antibody levels in immunized pigs. \u003c/strong\u003eSPF pigs were primed with either VSV*DG (groups A and C) or the VSVDG(H1) + VSVDG(N1) cocktail (groups B and D) and subsequently boosted with either VSV*DG (group A), VSVDG(H1) + VSVDG(N1) (group B) or the genetically modified LAIV (groups C and D). Serum was prepared prior to vaccination (d0), 4 weeks after the primary vaccination (d28), and 4 weeks after the second immunization (d56). \u003cstrong\u003ea, b\u003c/strong\u003e The virus-neutralization dose 50% (ND\u003csub\u003e50\u003c/sub\u003e) was determined against either H1N1\u003csub\u003eHH4/09\u003c/sub\u003e (\u003cstrong\u003ea\u003c/strong\u003e) or H1N1\u003csub\u003eHH4/09\u003c/sub\u003e/H1N1\u003csub\u003eVic/19\u003c/sub\u003e 6:2 reassortant virus encoding the HA and NA antigens of H1N1\u003csub\u003eVic/19\u003c/sub\u003e (\u003cstrong\u003eb\u003c/strong\u003e). \u003cstrong\u003ec\u003c/strong\u003e The boost sera of groups A and B were tested for virus-neutralizing activity against H5N1\u003csub\u003eTex/24\u003c/sub\u003e. Sera from chickens (n = 5) that had been vaccinated with VSVDG(H5) encoding the HA of A/Dalmatian Pelican/Bern/1/2022 (H5N1) (a-H5) were used as positive control. Geometric mean values with 95% CI are shown. The two-way ANOVA test with Tukey`s multiple comparisons was used to identify significant ND\u003csub\u003e50\u003c/sub\u003e values between pre-immune (d0), prime (d28), and boost (d56) sera.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7818522/v1/e541b1a8b9ba00b536866d1d.png"},{"id":95523861,"identity":"d65e0991-f826-4570-a5a5-15f6e34d1520","added_by":"auto","created_at":"2025-11-10 10:01:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32966,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of sialidase\u003c/strong\u003e-\u003cstrong\u003einhibitory antibodies in sera of vaccinated pigs. \u003c/strong\u003eThe ELLA test was used to detect antibodies with inhibitory activity against the N1 sialidase of H5N1 reassortant viruses. \u003cstrong\u003ea\u003c/strong\u003e Preimmune (d0), prime (d28) and boost (d56) sera of the indicated vaccine groups were analyzed for inhibition of the N1\u003csub\u003eHH4/09\u003c/sub\u003e sialidase. \u003cstrong\u003eb\u003c/strong\u003e Prime (d28) and boost (d56) sera of group B pigs were analyzed with H5N1 reassortant viruses encoding the NA of either H1N1\u003csub\u003eHH4/09\u003c/sub\u003e, H1N1\u003csub\u003eVic/19\u003c/sub\u003e or H5N1\u003csub\u003eTex/24\u003c/sub\u003e, and H5N2\u003csub\u003eIba/05\u003c/sub\u003e. The inhibitory concentration 50% (IC\u003csub\u003e50\u003c/sub\u003e) for each of the individual sera, geometric mean values (height of the bars) with 95% CI are shown. The two-way ANOVA test with either Sidak`s or Tukey`s multiple comparisons was used to assess significant differences between prime and boost sera and to compare the inhibition of different N1 neuraminidases. Only significant differences (p \u0026lt; 0.05) are indicated.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7818522/v1/e29478dbbf3ff599ffc74e26.png"},{"id":95523943,"identity":"55ad28fe-2275-41f3-ad07-a386e2526d8d","added_by":"auto","created_at":"2025-11-10 10:01:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":101820,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibodies raised against N1\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eHH4/09\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e inhibit H5N1\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eTex/24\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e release from infected cells. \u003c/strong\u003eMDCK cells grown in 96-well cell culture plates were inoculated for 60 min with 100 TCID\u003csub\u003e50\u003c/sub\u003e/well of H5N1\u003csub\u003eTex/24\u003c/sub\u003e and subsequently incubated with MEM containing serially diluted boost serum pools from the indicated vaccine groups. \u003cstrong\u003ea, b\u003c/strong\u003e Cell culture supernatant was collected at 24 h (\u003cstrong\u003ea\u003c/strong\u003e) and 48 h (\u003cstrong\u003eb\u003c/strong\u003e) post infection and infectious virus titers determined. The lower limit of detection is indicated by a dotted line. \u003cstrong\u003ec\u003c/strong\u003e Determination of the inhibitory concentration 90% (IC\u003csub\u003e90\u003c/sub\u003e) of boost sera from individual animals of the indicated vaccine groups causing 90% reduction of H5N1\u003csub\u003eTex/24\u003c/sub\u003e or H5N4\u003csub\u003eSwi/21\u003c/sub\u003e release into the cell culture supernatant 24 h post infection. Only sera from group A and B animals were analyzed for inhibition of H5N4 release. The lower limit of detection is indicated by a dotted line. \u003cstrong\u003ed\u003c/strong\u003e At 48 h post infection with H5N1\u003csub\u003eTex/24\u003c/sub\u003e, cells were fixed and the influenza NP antigen detected by indirect immunofluorescence (green fluorescence). Nuclei were stained by DAPI (blue fluorescence). Mock-infected cells and infected cells incubated with group A and group B sera at the indicated dilutions are shown. Bar size = 100 mm.\u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7818522/v1/422ad921e140434d55ad67f8.png"},{"id":95524178,"identity":"19e87a2d-359e-4d15-942e-0ab17ace2bb9","added_by":"auto","created_at":"2025-11-10 10:02:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":38235,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of LAIV shedding. \u003c/strong\u003eFollowing primary immunization of group C and group D animals (n = 5 animals/group) with either VSV*DG (group C) or VSVDG(H1)/VSVDG(N1) cocktail (group D), the animals were boosted intranasally with the LAIV NS1(1-126)-DPAX. Sentinel animals (n = 3) were added to each group and co-housed with the LAIV boosted animals for three weeks. \u003cstrong\u003ea\u003c/strong\u003eRT-qPCR analysis of nasal swab samples collected at the indicated days post vaccination with LAIV. The 45-Ct is the cycle threshold number subtracted from the maximum number of cDNA amplification cycles. \u003cstrong\u003eb\u003c/strong\u003e Determination of the focus-forming units (FFU) in nasal swab samples suspended in 0.75 mL of MEM. The lower detection limit is indicated by a dotted line. \u003cstrong\u003ec\u003c/strong\u003e Analysis of seroconversion of pigs to the influenza NP antigen. Sera from group C and D animals were prepared four weeks after the booster vaccination (d56). Sera of the corresponding sentinel animals were prepared three weeks after they had been grouped with the vaccinated pigs (d49). NP-specific serum antibodies were detected by a competitive ELISA. The ratio of the optical density at 450 nm of sample to negative control (S/N%) was calculated. Group A animals that were immunized with the control vaccine VSV*DG served as negative control. A significant difference in S/N (%) indicating seroconversion was assessed by the one-way ANOVA test adjusted to Dunnett`s multiple comparisons and group A as reference.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7818522/v1/7229c5e09064dd84c69c49a6.png"},{"id":95328172,"identity":"12c71dcf-b307-4f15-84b2-b2150c3c35db","added_by":"auto","created_at":"2025-11-06 18:26:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":52870,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVirus loads in lung tissue and nasal swab samples.\u003c/strong\u003e Four weeks after the booster vaccination, SPF pigs were challenged via the nasal route with the H1N1\u003csub\u003eHH4/09\u003c/sub\u003e:H1N1\u003csub\u003eVic/19\u003c/sub\u003e 6:2 reassortant virus encoding the HA and NA antigens of H1N1\u003csub\u003eVic/19\u003c/sub\u003e. \u003cstrong\u003ea \u003c/strong\u003eDetection by RT-qPCR of viral RNA in nasal swab samples collected at the indicated days post infection. The cycle threshold (Ct) subtracted from 45, i.e. the maximum number of cycles performed, is shown. \u003cstrong\u003eb\u003c/strong\u003e Detection of infectious virus in nasal swab samples. Swab samples were suspended in 0.75 mL of MEM and infectious virus titrated on MDCK cells. The focus-forming units (FFU) are shown for all swabs collected from individual animals in the indicated groups. The lower detection limit is indicated by a dotted line. \u003cstrong\u003ec\u003c/strong\u003e Detection by RT-qPCR of viral RNA extracted from tonsils, mesenteric lymph nodes (MLN) tracheobronchial lymph nodes (TBLN), lung lobes (LL) of euthanized pigs at day 5 post infection.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7818522/v1/4c8332198e1f3c08be16f296.png"},{"id":105755669,"identity":"05a22dc3-0003-434a-8765-ee8d8afb2bb9","added_by":"auto","created_at":"2026-03-30 16:29:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1616616,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7818522/v1/d40c906c-8f0a-4968-a9d2-3adafe4e7ce6.pdf"},{"id":95328166,"identity":"12e559f5-e30a-4e77-a3c3-cc6c68298019","added_by":"auto","created_at":"2025-11-06 18:26:53","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":64848,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 1 | Recording of the rectal temperature in SPF pigs following vaccination and challenge infection\u003c/strong\u003e. \u003cstrong\u003ea-d\u003c/strong\u003e The rectal temperature was recorded in all animals of the indicated groups (n = 5 animals/group) at the indicated day of the experiment. Mean values with SD (dotted lines) are shown. \u003cstrong\u003ea\u003c/strong\u003eRecording of rectal temperature following prime vaccination of groups A - D. \u003cstrong\u003eb\u003c/strong\u003eRecording of the rectal temperature in animals of groups A and B following boost vaccination. C Recording of rectal temperature in animals of group C and D (n = 5/group) after receiving the LAIV and in the sentinal pigs ( n = 3) that were co-housed with each of the groups.\u003c/p\u003e","description":"","filename":"SupplementaryFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7818522/v1/335d9d12ad4ce773bb0a7603.png"},{"id":95328164,"identity":"c8e47213-c49f-412b-b60e-4d78ac2972c6","added_by":"auto","created_at":"2025-11-06 18:26:53","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12100,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 2 \u003c/strong\u003e| \u003cstrong\u003eAlignment of HA primary amino acid sequences\u003c/strong\u003e. The HA sequences of A/Hamburg/4/2009 (H1N1) (GenBank accession no. ACR10223) and A/Victoria/2570/2019 (H1N1) (GenBank accession no. WEY08940) are shown. The predicted signal sequences (amino acids 1-17) and transmembrane domains (amino acids 537-560) are depicted with a grey background. Amino acid positions differing between the two HA antigens are shown in bold letters. The amino acid identity of the two HA antigens is 95.5%.\u003c/p\u003e","description":"","filename":"SupplementaryFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7818522/v1/af3928c901c944c513ea253c.png"},{"id":95523857,"identity":"b588bbc2-d854-48ce-8a0c-8181f6e83dc8","added_by":"auto","created_at":"2025-11-10 10:01:15","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":11145,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 3 | Alignment of NA primary amino acids sequences. \u003c/strong\u003eAlignment of the NA amino acid sequences of A/Hamburg/4/2009 (H1N1) (GenBank accession no. ACR10227), A/Victoria/2570/2019 (H1N1) (GenBank accession no. WEY08939), and A/cattle/Texas/063224-24-1/2024 (H5N1) (GISAID acc. no. EPI_ISL_19155861) are shown. The predicted transmembrane domains (amino acids 7-30) are depicted with a grey background. Amino acid positions differing between the two NA antigens are shown in bold letters.\u003c/p\u003e","description":"","filename":"SupplementaryFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7818522/v1/2aacb9a34bd9777f713bdeb9.png"},{"id":95524077,"identity":"74c4c218-647a-46eb-9232-05abba1355e6","added_by":"auto","created_at":"2025-11-10 10:02:08","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":27070,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7818522/v1/a7b596e140a33f0826986556.docx"}],"financialInterests":"Competing interest reported. RA, GZ, and AS filed a patent application related to the intramuscular prime/intranasal boost vaccine described in this work. All other authors declare no competing interests.","formattedTitle":"An RNA replicon vaccine encoding HA and NA prevents shedding of antigen- drifted 2009 pandemic H1N1 influenza virus in the pig model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInfluenza A viruses (IAV) encode two important envelope glycoproteins that are targeted by the immune system of the host. The hemagglutinin (HA) is a trimeric transmembrane protein with a large ectodomain that is structured into a variable globular head domain and a more conserved stalk region. The globular head domain harbors the receptor-binding pocket that interacts with sialic acid residues on cellular glycoproteins and glycolipids \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Most virus-neutralizing antibodies that are produced by the immune system in response to IAV infection are directed to the globular head and interfere with the receptor-binding activity of HA \u003csup\u003e1,2\u003c/sup\u003e. However, IAV are highly mutable, and the immune response frequently selects for viral mutants that are characterized by amino acid substitutions in the globular head region, which prevents binding of neutralizing antibodies, a phenomenon known as \u0026ldquo;antigenic drift\u0026rdquo; \u003csup\u003e3\u003c/sup\u003e. Because of this antigenic drift, seasonal influenza vaccines need to be updated every year according to WHO recommendations which are published about six months before the upcoming influenza season. Sometimes this prediction is inaccurate and a mismatch between the vaccine strain and presently circulating seasonal IAV leads to low vaccine efficacy \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Thus, there is a need for vaccines that provide efficient protection against antigen-drifted IAV.\u003c/p\u003e\u003cp\u003eNeuraminidase (NA) is the second important antigen of IAV. The tetrameric transmembrane protein exhibits sialidase activity which is essentially required for release of progeny virus from the infected cell membrane \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, as well as for penetration of the mucus-rich layers covering the respiratory epithelia of the host \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Antibodies that inhibit NA sialidase activity can efficiently interfere with virus dissemination and thus have protective potential \u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Not surprisingly, NA is also subject to antigenic drift \u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Of note, the most used vaccines to control seasonal influenza epidemics are inactivated split IAV standardized for hemagglutinin (HA) content, whereas the protective features of the NA are usually neglected \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn our previous work, we developed a novel prime/boost vaccination strategy comprising of an intramuscular prime with recombinant RNA replicon particles composed of G protein-deleted vesicular stomatitis virus (VSVΔG) vector encoding the H1 antigen of the pandemic A/Hamburg/4/2009 (H1N1) strain (H1N1\u003csub\u003eHH4/09\u003c/sub\u003e) followed by an intranasal boost using a live-attenuated influenza vaccine (LAIV) based on a modified H1N1\u003csub\u003eHH4/09\u003c/sub\u003e characterized by a truncated NS1 protein and lack of PA-X protein expression (NS1(1-126)-ΔPAX) \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. This prime/boost vaccination protocol elicited a strong systemic and mucosal immune response in the porcine model and resulted in sterilizing immunity against challenge infection with the homotypic H1N1\u003csub\u003eHH4/09\u003c/sub\u003e strain. In addition, priming with H1-recombinant RNA replicon particles controls replication and shedding of LAIV, thereby adding to vaccine safety \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn the present study, we investigated the efficacy of the heterologous RNA replicon prime/LAIV boost strategy in protecting pigs against antigen-drifted H1N1 virus and compared it with a homologous RNA replicon prime/RNA replicon boost strategy. To achieve a broader immune response, a cocktail of two VSV-based RNA replicon particles was used for intramuscular priming of the animals, VSVΔG(H1) and VSVΔG(N1), which encode the HA and NA antigens of H1N1\u003csub\u003eHH4/09\u003c/sub\u003e, respectively. In the homologous prime/boost strategy, the animals were immunized a second time with the RNA replicon particles, while in the heterologous strategy, they were immunized intranasally with LAIV NS1(1-126)-ΔPAX. The immune response of the animals was examined for H1-specific virus-neutralizing antibodies as well as N1-specific antibodies showing sialidase-inhibitory activity. Finally, the animals were challenged with a reassortant virus encoding the HA and NA antigens of the antigen-drifted A/Victoria/2570/2019 (H1N1) (H1N1\u003csub\u003eVic/19\u003c/sub\u003e) strain. Our results suggest that while both prime/boost strategies efficiently controlled replication and nasal shedding of the antigen-drifted virus, the robust N1-specific response induced by the VSV replicons turned out to be broadly protective, also inhibiting zoonotic H5N1 virus isolated from infected dairy cows.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eEvaluation of homologous and heterologous prime/boost vaccination strategies in the pig animal model\u003c/h2\u003e\u003cp\u003eSpecific pathogen-free (SPF) pigs (8 to 10 weeks of age) were randomly distributed into 4 groups with 5 animals each (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Animals of groups A and C were first immunized via the intramuscular (i.m.) route with VSV*ΔG, RNA replicon particles encoding the irrelevant GFP protein, while animals of groups C and D were immunized (i.m.) with VSVΔG(H1) and VSVΔG(N1) replicon particles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Four weeks after the primary immunization, the pigs were boosted with either VSV*ΔG (group A) or VSVΔG(H1) and VSVΔG(N1) replicon particles (group B), while animals of groups C and D were boosted via the intranasal route with the LAIV NS1(1-126)-ΔPAX \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Vaccination of the animals with VSV replicon particles or LAIV was well tolerated by the animals and did not cause any significant clinical symptoms (\u003cb\u003eSupplementary Fig.\u0026nbsp;1, Supplementary Table\u0026nbsp;1\u003c/b\u003e). Nasal swab samples were collected on the following days from the animals of groups C and D to monitor shedding of the LAIV. In addition, three immunologically na\u0026iuml;ve pigs were added to each group C and group D two days after the booster vaccination to monitor potential transmission of the virus to the sentinel animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFour weeks after the second vaccination, the pigs were infected via the intranasal route with a reassortant virus containing the genomic segments 4 (HA) and 6 (NA) of A/Victoria/2570/2019 (H1N1) (H1N1\u003csub\u003eVic/19\u003c/sub\u003e) and the remaining 6 genomic segments of A/Hamburg/4/2009 (H1N1) (H1N1\u003csub\u003eHH4/09\u003c/sub\u003e). The Victoria strain has been recommended as a component of the tetravalent influenza vaccine for use in the 2021 and 2022 southern hemisphere seasons \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e and for the 2022/2023 season in the northern hemisphere \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Compared to the original 2009 pandemic H1N1 virus, the HA and NA antigens of this 2019 H1N1 virus were characterized by several amino acid substitutions in their ectodomains (\u003cb\u003eSupplementary Figs.\u0026nbsp;2, 3\u003c/b\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eThe second immunization boosts the neutralizing antibody response against antigen-drifted H1N1 virus\u003c/h3\u003e\n\u003cp\u003eTo monitor H1N1-specific antibody responses, serum samples were prepared prior to vaccination (d0, pre-immune serum), 4 weeks after the primary vaccination (d28, prime serum) and 4 weeks after the second immunization (d56, boost serum) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and analyzed for virus-neutralizing activity against H1N1\u003csub\u003eHH4/09\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) and the H1N1\u003csub\u003eHH4/09\u003c/sub\u003e/H1N1\u003csub\u003eVic/19\u003c/sub\u003e 6:2 reassortant virus encoding the segments 4 and 6 of H1N1\u003csub\u003eVic/19\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). None of the SPF pigs had pre-existing neutralizing antibodies to either of the two viruses. Pigs of group A that had been immunized with the control vaccine VSV*ΔG did not develop any H1N1-neutralizing antibodies, as expected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). In contrast, animals of group B developed significantly high titers of H1N1\u003csub\u003eHH4/09\u003c/sub\u003e-neutralizing antibodies (geometric mean ND\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;485) after the first immunization with VSVΔG(H1) and VSVΔG(N1) replicon particles. The ND\u003csub\u003e50\u003c/sub\u003e titer increased further following the second immunization with the same vaccine (geom. mean ND\u003csub\u003e50\u003c/sub\u003e titer\u0026thinsp;=\u0026thinsp;1358), however, the difference between the ND\u003csub\u003e50\u003c/sub\u003e titers of the prime and boost sera was not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). When the immune sera of group B were tested for neutralizing activity against the H1N1\u003csub\u003eHH4/09\u003c/sub\u003e/H1N1\u003csub\u003eVic/19\u003c/sub\u003e 6:2 reassortant virus, the prime sera showed only very low neutralizing activity (geom. mean ND\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6), which increased significantly after the second administration of the vaccine (geom. mean ND\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;277) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). When pigs were first immunized (i.m.) with VSV*ΔG and subsequently with the LAIV via the intranasal route (group C, d56), only low H1N1\u003csub\u003eHH4/09\u003c/sub\u003e-neutralizing serum antibody titers were detected (geom. mean ND\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;30) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), and inhibitory activity against the H1N1\u003csub\u003eHH4/09\u003c/sub\u003e/H1N1\u003csub\u003eVic/19\u003c/sub\u003e 6:2 reassortant virus was even lower (geom. mean ND\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). However, when the animals were first primed (i.m.) with VSVΔG(H1) and VSVΔG(N1) and then intranasally immunized with the LAIV (group D), geometric mean ND\u003csub\u003e50\u003c/sub\u003e values against the antigen-drifted H1N1\u003csub\u003eHH4/09\u003c/sub\u003e/H1N1\u003csub\u003eVic/19\u003c/sub\u003e 6:2 reassortant virus increased from 6 (d28, after prime) to 276 (d56, after boost) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), while neutralizing activity against the homotypic H1N1\u003csub\u003eHH4/09\u003c/sub\u003e strain was already high after the first immunization (geom. mean ND\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;365), and increased only marginally following immunization with LAIV (geom. mean ND\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;958) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Finally, we tested whether the boost sera of group B would show some neutralizing activity against A/cattle/Texas/063224-24-1/2024 (H5N1) (H5N1\u003csub\u003eTex/24\u003c/sub\u003e), however, no significant inhibition of this virus was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). In conclusion, our findings suggest that a prime/boost vaccination regimen is important to produce robust levels of serum antibodies with neutralizing activity against antigen-drifted H1N1 virus.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eVaccinated pigs produce N1-specific antibodies with sialidase-inhibitory activity\u003c/h3\u003e\n\u003cp\u003eTo measure the functional antibody response triggered by the VSVΔG(N1) vaccine component, we performed an enzyme-linked lectin assay (ELLA) to detect antibodies inhibiting the enzyme activity of the N1 neuraminidase. The assay is based on immobilized fetuin as substrate and biotin-labeled peanut agglutinin (PNA) as probe for the desialylated product of the enzyme reaction \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Since the immune sera of immunized pigs also contain H1-specific antibodies that could sterically block the binding of N1-specific antibodies to the virion \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, the test was performed with H5N1\u003csub\u003eHH4/09\u003c/sub\u003e, a bovine H5N1 virus with its genome segment 6 (NA) replaced by the corresponding segment of H1N1\u003csub\u003eHH4/09\u003c/sub\u003e. For biosafety reasons, the proteolytic cleavage site in the H5 hemagglutinin was converted into a monobasic motif as found in low-pathogenic H5Nx influenza viruses \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Pigs that had been vaccinated with the VSV*ΔG control vaccine (group A) did not develop antibodies with inhibitory activity against N1\u003csub\u003eHH4/09\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Likewise, pigs immunized via the intranasal route with the LAIV (group C) did not develop significant levels of serum antibodies with N1 inhibitory activity. In contrast, pigs developed geometric mean sialidase inhibitory antibody titers of IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1267 (group B) and IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1283 (group D) after the prime immunization (i.m.) with the VSVΔG(H1) plus VSVΔG(N1) vaccine. The second intramuscular administration of the vaccine significantly boosted the N1-specific antibody response in group B (geom. mean IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7098), while the intranasal boost with the LAIV (group D) did not result in significantly increased inhibitory antibody titers (geom. mean IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2065).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo analyze the inhibitory activity of the immune sera against N1 sialidases from other influenza viruses, we also generated H5N1 reassortant viruses encoding the N1 sialidases of either H5N1\u003csub\u003eVic/19\u003c/sub\u003e or H5N1\u003csub\u003eTex/24\u003c/sub\u003e and the N2 sialidase of A/chicken/Ibaraki/1/2005 (H5N2) (H5N2\u003csub\u003eIba/05)\u003c/sub\u003e. Interestingly, the immune sera from group B pigs showed similarly high inhibitory activity against all three N1 sialidases but did not inhibit the N2 sialidase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). These results show that the intramuscular immunization with the VSVΔG(N1) vaccine component led to the production of N1-specific serum antibodies that have potent sialidase inhibitory activity, even against distantly related N1 such as the one from the bovine H5N1\u003csub\u003eTex/24\u003c/sub\u003e virus.\u003c/p\u003e\n\u003ch3\u003eAntibodies to the N1 neuraminidase inhibit the release of H5N1 from infected MDCK cells\u003c/h3\u003e\n\u003cp\u003eImmune sera from vaccinated pigs of group B did not show neutralizing activity against H5N1\u003csub\u003eTex/24\u003c/sub\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) but showed potent inhibition of N1\u003csub\u003eTex/24\u003c/sub\u003e sialidase activity (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). To study the impact of N1 sialidase inhibition on the release of infectious H5N1\u003csub\u003eTex/24\u003c/sub\u003e, we infected 96-well MDCK cell cultures with 100 TCID\u003csub\u003e50\u003c/sub\u003e/well of H5N1\u003csub\u003eTex/24\u003c/sub\u003e and then added cell culture medium containing serially diluted immune sera. These serum pools were prepared for all four vaccine groups four weeks after the second immunization. At 24 and 48 hours p.i., the infectious titers of H5N1\u003csub\u003eTex/24\u003c/sub\u003e released into the cell culture supernatants were determined on MDCK cells by end point titration. Immune sera of groups A and C did not inhibit the release of H5N1\u003csub\u003eTex/24\u003c/sub\u003e from infected MDCK cells, independently of the serum concentration used (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). In contrast, immune sera from group B showed a concentration-dependent inhibition of H5N1\u003csub\u003eTex/24\u003c/sub\u003e release. Using the immune serum at a dilution of 1:20, a remarkable reduction of infectious titer by 6 log\u003csub\u003e10\u003c/sub\u003e was achieved at 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), which was still present at 48 h p.i. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Likewise, pooled group D immune serum mediated concentration-dependent inhibition of H5N1\u003csub\u003eTex/24\u003c/sub\u003e release, however, with general lower efficacy compared to group B immune serum. Analysis of the sera from individual animals allowed us to determine the serum concentration leading to 90% inhibition of H5N1\u003csub\u003eTex/24\u003c/sub\u003e release at 24 hours p.i. \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec\u003cb\u003e).\u003c/b\u003e The geometric mean IC\u003csub\u003e90\u003c/sub\u003e titer was 896 for group B and 160 for group D, whereas group A and C sera had no significant impact on virus release. Release of a HPAI H5N4 virus was not inhibited by the group B serum pool, indicating that the inhibitory activity of the immune sera is NA subtype specific. At 48 h p.i., when the cell culture supernatant had been removed, the cells were fixed with formalin and infected cells visualized by indirect immunofluorescence using a monoclonal antibody directed to the influenza NP antigen. In MDCK cells incubated with group A or group C serum, H5N1\u003csub\u003eTex/24\u003c/sub\u003e infection spread throughout the whole monolayer, whereas in cells incubated with group B or D serum the infection remained restricted to distinct foci (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Collectively, these results demonstrate that the VSVΔG(N1) vaccine component induces N1-specific serum antibodies with potent inhibitory activity against H5N1\u003csub\u003eTex/24\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eIntramuscular priming of pigs eliminates LAIV shedding\u003c/h3\u003e\n\u003cp\u003eIn a recent study we showed that the prolonged shedding of the LAIV NS1(1-126)-ΔPAX from the upper respiratory tract of vaccinated pigs can be reduced if the animals were first primed with VSVΔG(H1) replicon particles \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. To confirm this finding and to see whether the vaccination of the pigs with VSVΔG(H1) in combination with VSVΔG(N1) could completely eliminate LAIV shedding, the vaccinated pigs of groups C and D were monitored for a total of 16 days by analyzing nasal swab samples by RT-qPCR for the presence of viral RNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) and infectious virus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Using these methods, we detected significant LAIV shedding by animals of group C, which lasted for several days. The three non-vaccinated pigs that were kept for three weeks with the animals of group C seroconverted to the influenza NP antigen (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), indicating that LAIV was transmitted to these sentinels. In contrast, if the pigs were first primed with the VSVΔG(H1)/VSVΔG(N1) vaccine, only some viral RNA was detected in nasal swabs at day 1 and 2 after nasal administration of the LAIV (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), and no shedding of infectious virus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) or transmission to sentinels was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). These data confirmed that intramuscular priming with the H1/N1 RNA replicon particles efficiently controlled replication of the LAIV in the upper respiratory tract.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eVaccination of pigs efficiently prevents replication of antigen-drifted H1N1 virus in the respiratory tract\u003c/h2\u003e\u003cp\u003eFour weeks after the second vaccination, the animals were challenged via the intranasal route with 10\u003csup\u003e6\u003c/sup\u003e infectious units/pig of the H1N1\u003csub\u003eHH4/09\u003c/sub\u003e/H1N1\u003csub\u003eVic/19\u003c/sub\u003e 6:2 reassortant virus. None of the animals, including those in control group A, showed an elevated rectal temperature (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e) or other clinical symptoms (\u003cb\u003eSupplementary Table\u0026nbsp;2\u003c/b\u003e) as a result of the infection. To monitor the shedding of challenge virus, nasal swab samples were collected daily for a total of 5 days post infection and analyzed for the presence of viral RNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) and infectious virus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). As expected, all animals of control group A displayed shedding of challenge virus starting from day 2 and lasting until the end of the experiment at day 5 post infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). In contrast, no viral RNA was detected in swab samples taken from group C animals at any of the post challenge days, and only some swab samples collected from pigs of groups B and D were positive for viral RNA on some days (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). However, no infectious virus was detected in the nasal swabs collected from the pigs from groups B, C, and D (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In line with these findings, viral RNA was detected in tonsils, mesenteric and tracheobronchial lymph nodes and lung lobes in most animals of group A but was essentially absent from the corresponding tissues of animals of group B, C, and D \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec\u003cb\u003e).\u003c/b\u003e These results show that both the heterologous and homologous prime/boost vaccination regimens are effective in preventing the replication and shedding of antigen-drifted virus.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eHeterologous prime/boost immunization\u003c/h2\u003e\u003cp\u003eAntigen-drift represents a major hurdle for the efficacy of conventional influenza vaccines that rely on inactivated split viruses and are standardized to the amount of purified HA antigen. In the present work, we show that intranasal immunization of pigs (group C) with the LAIV NS1(1-126)-ΔPAX triggers an immune response that efficiently controlled the replication of a reassortant virus bearing the HA and NA envelope glycoproteins of the antigen-drifted H1N1\u003csub\u003eVic/19\u003c/sub\u003e strain. Although intranasal immunization with LAIV induced relatively low levels of virus-neutralizing serum antibodies, previous work with the same LAIV showed that intranasal immunization of pigs with this vaccine induces local immune responses including secretion of H1N1-specific IgA into the mucosal tissues and cellular immune responses that are expected to contribute to its potent inhibition of challenge virus replication in the mucosa \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. However, the prolonged shedding of LAIV via the respiratory tract and transmission to sentinel pigs was identified as a safety issue \u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e–\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. This problem was solved by first immunizing the animals via the intramuscular route with VSV-based RNA replicon particles encoding HA \u003csup\u003e22\u003c/sup\u003e. This immune priming induced high levels of systemic immunity that efficiently controlled LAIV shedding without preventing the LAIV from boosting neutralizing antibodies. In the present study, immune priming with both H1 and N1 antigen also eliminated LAIV shedding. Still, the boost vaccination using the LAIV significantly enhanced the systemic immune response against the antigen-drifted HA\u003csub\u003eVic/19\u003c/sub\u003e protein. Furthermore, the inclusion of VSV replicons encoding the N1\u003csub\u003eHH4/09\u003c/sub\u003e antigen resulted in the induction antibodies that inhibited the sialidase activity of N1\u003csub\u003eHH4/09\u003c/sub\u003e and N1\u003csub\u003eVic/19\u003c/sub\u003e equally well. As a result, this vaccination protocol induced robust cross-protective immunity, even preventing shedding of heterologous challenge virus from the respiratory tract.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eHomologous prime/boost immunization\u003c/h2\u003e\u003cp\u003eThe first immunization (i.m.) with the combined VSVΔG(H1) and VSVΔG(N1) vaccine resulted in high antibody titers with neutralizing activity against homologous H1N1\u003csub\u003eHH4/09,\u003c/sub\u003e which increased only moderately after administration of the second vaccine dose. In contrast, the neutralizing activity against antigenically drifted H1N1\u003csub\u003eVic/19\u003c/sub\u003e strain was strongly boosted by the second dose. This broadened immunity was likely driven by memory B cells undergoing affinity maturation upon antigen re-exposure \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Notably, re-stimulating memory B cells with the same antigens enhances not only the strength and quality but also the longevity of antibody responses \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAnother important finding of our study was that homologous prime/boost strategy using the combined replicon particle vaccines eliminated the shedding of the antigen-drifted H1N1 challenge virus, even though the vaccines were administered via the intramuscular route. \u003csup\u003e28\u003c/sup\u003e. Possibly, H1- and N1-specific serum IgG was secreted into the mucosal tissues of the respiratory tract via the neonatal Fc receptor (FcRn) which is expressed in mammalian species also at adult life \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In mice, passive intravenous transfer of anti-influenza immunoglobulins indicated that high serum IgG levels can efficiently reduce nasal influenza virus shedding \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, suggesting that a fraction of the serum IgG can be secreted to mucosal surfaces. Thus, the strong immune response induced by intramuscular immunization with the RNA replicon particles is the likely key factor preventing challenge virus replicating in the respiratory mucosa and, consequently, virus shedding.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eImportance of NA-specific antibodies in protection\u003c/h2\u003e\u003cp\u003eIn contrast to our previous study in which the VSVΔG(H1) vaccine was used for immunization of pigs \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, the present work made also use of VSVΔG(N1) replicon particles encoding N1\u003csub\u003eHH4/09\u003c/sub\u003e. The N1-specific antibodies elicited by this vaccine potently inhibited the sialidase activity of the homologous N1\u003csub\u003eHH4/09\u003c/sub\u003e but also the sialidase activity of N1\u003csub\u003eVic/19\u003c/sub\u003e and N1\u003csub\u003eTex/24\u003c/sub\u003e, although the latter shows only 88% amino acid identity with N1\u003csub\u003eHH4/09\u003c/sub\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;2\u003c/b\u003e). This suggests that the N1-specific antibodies bound to epitopes that are conserved in this antigen. There is evidence that the antigenic drift associated with NA is slower than that of the HA antigen, possibly because of functional constraints, i.e. only mutations that do not significantly impair sialidase activity are tolerated \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eNA sialidase activity is crucial for the efficient release of progeny influenza virus from the infected host cell \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e but also helps the virus to penetrate the mucus layers covering the epithelial cells of the respiratory tract \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Thus, the antibodies induced by VSVΔG(N1) likely contributed to immune protection of the pigs by inhibiting virus release and spread. Indeed, previous work has shown that VSVΔG(NA) replicon particles are protective even if used as a stand-alone vaccine \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. The observation that immune sera of pigs immunized with the combined VSVΔG(H1) and VSVΔG(N1) replicon particles potently inhibited release and spread of H5N1\u003csub\u003eTex/24\u003c/sub\u003e, despite lacking virus-neutralizing activity, underscores the enhanced broad cross-reactivity achieved by targeting NA in vaccination.\u003c/p\u003e\u003cp\u003eIn recent years, H5N1 HPAI viruses of the clade 2.3.4.4b have spread worldwide and led to the death of millions of birds of numerous species \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In addition, clade 2.3.4.4b viruses showed the remarkable potential to infect various mammalian species \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, including humans \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. While infection of cats by H5N1 caused neurological symptoms with often fatal outcome \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, infection of humans was mostly associated with mild symptoms such as conjunctivitis \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. There is evidence that infection with human influenza viruses elicits broadly cross-reactive NA-specific antibodies that protect from infection by HxN1 viruses \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The protective effect of prior infection with 2009 pandemic H1N1 virus on the pathogenesis and transmission of clade 2.3.4.4b H5N1 virus was also observed in the ferret model \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. These findings imply that children who have not experienced infection with seasonal H1N1 virus yet might be at higher risk to develop severe symptoms if infected with H5N1 HPAI. Indeed, severe disease following infection with clade 2.3.4.4b genotype D1.1 was documented for a 13-year-old teenager in 2024 in Canada \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e and a fatal infection of a 3-year-old girl was reported in 2025 in Mexico \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. However, it is not known whether these children were more susceptible to H5N1 infection because they had no pre-existing immunity to influenza viruses in general.\u003c/p\u003e\u003cp\u003eTaken together, the present study not only confirms the efficacy and safety of influenza vaccines based on non-replicating RNA replicon particles in the porcine animal model \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, but demonstrates that the high levels of neutralizing anti-HA as well as the NA-targeting antibodies ensure broad systemic and mucosal protection against antigen-drifted influenza viruses. These properties represent a significant improvement compared to currently used inactivated seasonal influenza vaccines \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Methods","content":"\u003ch2\u003eCells\u003c/h2\u003e\u003cp\u003eMadin-Darby canine kidney type II cells (MDCK-II) were kindly provided by Georg Herrler (University of Veterinary Medicine, Hannover, Germany) and maintained with minimum essential medium (MEM, Thermo Fisher Scientific, Basel, Switzerland; cat. no. 31095-029) supplemented with 5% of fetal bovine serum (FBS; Pan Biotech, Aidenbach, Germany; cat. no. P30-3033). Human embryonic kidney (HEK) 293T cells (American Type Culture Collection (ATCC), Manassas, USA; cat. no. CRL-3216) were maintained with Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific; cat. no. 32430-027) supplemented with 10% FBS. Baby hamster kidney 21 (BHK-21) fibroblasts were obtained from ATCC (cat. no. CCL-10) and maintained in Glasgow’s minimal essential medium (GMEM, Thermo Fisher Scientific, cat. no. 21710-025) supplemented with 5% FBS. BHK-G43 cells, a transgenic BHK-21 cell clone expressing VSV glycoprotein G in a regulated manner \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, was maintained in GMEM supplemented with 5% FBS. All cells were grown at 37°C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere.\u003c/p\u003e\u003ch2\u003eViruses\u003c/h2\u003e\u003cp\u003eThe HPAI virus A/cattle/Texas/063224-24-1/2024 (H5N1) (H5N1\u003csub\u003eTex/24\u003c/sub\u003e), clade 2.3.4.4b, genotype B3.13 (GISAID accession number: EPI_ISL_19155861), was kindly provided by Diego Diel (Cornell University, Ithaca, NY, USA). This virus was originally isolated from the milk of infected dairy cows in Texas, USA \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. A/Buzzard/Switzerland/V0052/2021 (H5N4) (H5N4\u003csub\u003eSwi/21\u003c/sub\u003e) was isolated by IVI Mittelhäusern from a brain biopsy sample taken from a common buzzard that was found dead close to Lake Constance in 2021. The 2009 pandemic virus strain A/Hamburg/4/2009 (H1N1) (H1N1\u003csub\u003eHH4/09\u003c/sub\u003e) was produced by reverse genetics using the 8-plasmid system \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The live-attenuated influenza vaccine (LAIV) NS1(1-126)-ΔPAX is based on a modified H1N1\u003csub\u003eHH4/09\u003c/sub\u003e and encodes a truncated NS1 protein and is unable to drive expression of the accessory PA-X protein \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. VSV*ΔG is a propagation-defective vesicular stomatitis virus in which the glycoprotein (G) gene has been replaced by the green fluorescent protein (GFP) gene \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003ch2\u003eGeneration of recombinant influenza viruses\u003c/h2\u003e\u003cp\u003eThe cDNAs encoding the genomic segments 4 and 6 of A/Victoria/2570/2019 (H1N1) (GenBank acc. nos. WEY08940 and WEY08939, respectively) and the cDNAs of the eight genomic segments of A/bovine/Texas/24-029328-02/2024 (H5N1) (GenBank accession nos. PP599470–PP599477) \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e were synthesized by Genscript Biotech (Piscataway, New Jersey, USA). The synthetic cDNA of RNA segment 4 encoded an HA containing the monobasic cleavage site P\u003csub\u003e337\u003c/sub\u003eLRETR↓GLF rather than the polybasic cleavage site P\u003csub\u003e337\u003c/sub\u003eLREKRRKR↓GLF found in the authentic virus. All cDNAs were amplified with Phusion DNA and cloned into the \u003cem\u003eBsm\u003c/em\u003eBI restriction site of the pHW2000 plasmid \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e using the In-Fusion cloning system (Takara Bio, Saint-Germain-en-Laye, France, cat. no. 638948). The primary nucleotide sequence of each of the cloned segments was verified by Sanger sequencing.\u003c/p\u003e\u003cp\u003eInfectious viruses were generated by transfection of HEK 293T/MDCK cell co-cultures using the respective eight genomic cDNAs cloned into the pHW2000 vector and Lipofectamine 2000 (Life Technologies, cat. no. 11668019) as transfection reagent. In this way, the following recombinant viruses were generated: A virus containing segments 4 and 6 of H1N1\u003csub\u003eVic/19\u003c/sub\u003e and the remaining six segments of H1N1\u003csub\u003eHH4/09\u003c/sub\u003e, a H5N1\u003csub\u003eTex/24\u003c/sub\u003e with a monobasic proteolytic cleavage site in HA, and three reassortant viruses encoding segment 6 of either H1N1\u003csub\u003eHH4/09\u003c/sub\u003e, H1N1\u003csub\u003eVic/19\u003c/sub\u003e, or H5N1\u003csub\u003eTex/24\u003c/sub\u003e and the remaining genomic RNA segments of (H5\u003csub\u003emb\u003c/sub\u003eN1)\u003csub\u003eTex/24\u003c/sub\u003e. The rescued viruses were passaged two times on MDCK cells with FBS-deficient medium containing 1% penicillin/streptomycin and 1 µg/mL of acetylated trypsin (Merck KGaA, Darmstadt, Germany, cat. no. 4370285). Virus stocks were stored in aliquots at -70°C in the presence of 5% FBS.\u003c/p\u003e\u003ch2\u003eTitration of influenza A viruses\u003c/h2\u003e\u003cp\u003eFor titration of IAV that had a monobasic cleavage site in their HA, MDCK cells grown in 96-well tissue culture plates were inoculated in duplicate with 40 µL per well of serial 10-fold virus dilutions for 1 h at 37°C. Thereafter, 160 µL of MEM containing 1% methylcellulose were added to each well, and the cells incubated for 24 h at 37°C. The cells were fixed for 30 min with 4% formalin in PBS, permeabilized with 0.25% (v/v) of Triton X-100, and incubated for 60 min with a monoclonal antibody directed to the influenza nucleoprotein (NP) antigen (American Type Culture Collection, Manassas, Virginia, USA, ATCC HB-65, clone H16-L10-4R5, diluted 1:50 in PBS), and subsequently for 60 min with goat anti-mouse IgG conjugated with Alexa Fluor-488 (Life Technologies, Zug, Switzerland; diluted 1:500 in PBS). Infected cells were detected by fluorescence microscopy (Observer Z1 microscope, Zeiss, Feldbach, Switzerland), and infectious virus titers were calculated and expressed as focus-forming units per milliliter (ffu/mL).\u003c/p\u003e\u003cp\u003eFor titration of the HPAI H5N1\u003csub\u003eTex/24\u003c/sub\u003e, MDCK cell monolayers in 96-well tissue culture plates were incubated in quadruplicates with serially diluted virus (100 µL/well) for 48 h at 37°C. The cells were washed once with PBS (200 µL/well) and fixed for 1 h at 21°C with 4% of buffered formalin containing 0.1% (w/v) crystal violet (Merck KGaA, cat. no. 1.01408). The plates were washed with tap water and dried. Virus titer was calculated using the Spearman-Kärber method and expressed as tissue culture infectious dose 50% (TCID\u003csub\u003e50\u003c/sub\u003e) \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003ch2\u003eGeneration of recombinant VSV vector vaccine\u003c/h2\u003e\u003cp\u003eFor generation of the VSVΔG(H1) and VSVΔG(N1) vaccine components, the HA and NA genes of H1N1\u003csub\u003eHH4/09\u003c/sub\u003e (GenBank acc. nos. GQ166213 and GQ166217, respectively) were amplified by PCR using the Phusion DNA polymerase and inserted into the \u003cem\u003eMlu\u003c/em\u003eI and \u003cem\u003eNhe\u003c/em\u003eI sites of the pVSV*ΔG(HA\u003csub\u003eH5−HP\u003c/sub\u003e) plasmid \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, resulting in the pVSVΔG(H1) and pVSVΔG(N1) plasmids, respectively. VSVΔG(H1) and VSVΔG(N1) replicon particles were generated from transfected cDNA \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e and propagated on BHK-G43 cells expressing the VSV G protein in a regulated manner \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Virus stocks were stored in aliquots at -70°C. Infectious virus titers were determined on BHK-21 cells by end point dilution. Infected cells were detected 20 h p.i. by indirect immunofluorescence using a monoclonal antibody (mAb 23H12) directed to the VSV matrix protein (KeraFast, Boston, MA, cat. no. EB0011) \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFor production of vaccine batches, BHK-G43 cells were seeded into five T150 flasks and maintained in 40 mL/flask of GMEM medium with 5% FBS. When confluency was reached, the medium was aspirated and the cells maintained at 37°C for 6 h with 40 mL/flask of GMEM containing 10\u003csup\u003e− 9\u003c/sup\u003e M of mifepristone. VSVΔG(H5\u003csub\u003emb\u003c/sub\u003e) was added at a multiplicity of infection (moi) of 0.05 ffu/cell and incubated with the cells for 20 h at 37°C. The cell culture supernatant was transferred to 50-mL Falcon tubes and cell debris removed by centrifugation (1000 × g, 15 min, 4°C). The replicon particles were pelleted from the cleared supernatant by ultracentrifugation (105`000 × g, 60 min, 4°C) and resuspended in 20 mL of PBS. The VSVΔG(H1) and VSVΔG(N1) replicon particles were stored in aliquots at -70°C.\u003c/p\u003e\u003ch2\u003eAnimal experiments\u003c/h2\u003e\u003cp\u003e The animal experiments were performed according to Swiss laws (the Animal Welfare Act TSchG SR 455, the Animal Welfare Ordinance TSchV SR 455.1, and the Animal Experimentation Ordinance TVV SR 455.163). All experiments were reviewed by the committee on animal experiments of the canton of Bern and were approved by the cantonal veterinary authority under license number \u003cb\u003eBE15/2024\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eTwenty 10-week-old specific pathogen-free (SPF) Swiss Large White pigs from the breeding facility of the Institute of Virology and Immunology IVI (Mittelhäusern, Switzerland) were randomly divided into four groups (A – D), each containing five pigs of both sexes (n = 5). After an acclimatization period of five days, the animals of groups A and C received 2 mL of VSV*ΔG suspension (4 × 10\u003csup\u003e8\u003c/sup\u003e ffu/mL) that were injected into the \u003cem\u003em. brachiocephalicus\u003c/em\u003e (neck muscle) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The animals of groups B and D were immunized by injection of 2 mL (4 × 10\u003csup\u003e8\u003c/sup\u003e ffu/mL) of VSVΔG(H1) and 2 mL (4 × 10\u003csup\u003e8\u003c/sup\u003e ffu/mL) of VSVΔG(N1) suspension into different sites of the neck muscle. Body temperature and clinical symptoms were monitored daily for seven days, and serum samples were prepared at day 28 and day 56 after primary immunization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). At day 28, the animals of group A received a second dose (2 mL, 4 × 10\u003csup\u003e8\u003c/sup\u003e ffu/mL) of the control vector VSV*ΔG while animals of group B were immunized (i.m.) with VSVΔG(H1) and VSVΔG(N1) replicon particles using the same doses as for the first time. Animals of groups C and D were immunized via the nasal route with 5 mL of PBS containing 2 × 10\u003csup\u003e4\u003c/sup\u003e ffu/mL of LAIV. To this end, an intranasal mucosal atomization device (MAD Nasal, Teleflex Medical Europe Ltd., Ireland, cat. no, MAD300) plugged to a 10-mL syringe was employed.\u003c/p\u003e\u003cp\u003eAfter the first and the second immunization, the rectal temperature of the pigs and potential vaccine-mediated adverse effects were recorded daily for a total of 10 days. Blood (10 mL) was collected prior to vaccination (day 0), 28 days after prime vaccination, and 28 days after the booster immunization (day 56). Serum was prepared after coagulation of the blood and stored at -20°C prior to analysis by virus neutralization, sialidase inhibition and virus release inhibition tests. To monitor shedding of LAIV, two nasal swabs were taken from each animal of groups C and D prior to vaccination (day 28) and at days 29, 30, 32, 34, 36, 38, 40, 42, 44. One swab sample was soaked in 0.75 mL of MEM supplemented with penicillin/streptomycin (Life Technologies, cat. no. 15140-122) and amphotericin B (Life Technologies, cat. no. 15290018), while the second swab was soaked in 0.75 mL of RA1 lysis buffer (Macherey-Nagel, Düren, Germany; cat. no. 740961) containing 1% (v/v) β-mercaptoethanol. Swab-soaked media and lysates were stored at -70°C prior to analysis by virus titration and RT-qPCR, respectively. To monitor transmission of the LAIV, three sentinel animals were co-housed with each group C and group D at day 30 of the experiment (48 h after the second immunization). The sentinel animals were euthanized at day 54 and their serum tested for the presence of NP-specific antibodies by ELISA.\u003c/p\u003e\u003cp\u003eOn day 56, animals in groups A to D were infected intranasally with 2 ml PBS containing 10\u003csup\u003e6\u003c/sup\u003e ffu/ml of the H1N1\u003csub\u003eHH4/09\u003c/sub\u003e /H1N1\u003csub\u003eVic/19\u003c/sub\u003e 6:2 reassortant. After infection, rectal temperature and clinical symptoms, including liveliness, body tension, body shape, appetite, nasal discharge, respiratory rate, expiratory effort, wheezing/coughing and diarrhea, were recorded daily for a total of 5 days. A clinical scoring system was used to assess disease severity and determine the humane endpoint: 0 = no symptoms, 1 = mild symptoms, 2 = moderate symptoms, 3 = severe symptoms. The animals were euthanized immediately if they reached a total clinical score of 9, or if a clinical score of 3 was reached for one or more of the clinical parameters. In addition, nasal swab samples were taken daily and tested for the presence of viral RNA using RT-qPCR. Five days after infection, the animals were killed by electrocution and subsequent exsanguination. Immediately after exsanguination, biopsy material was taken from the tonsils, mesenteric lymph nodes, tracheobronchial lymph nodes and the right cranial lobe of the lung and tested for the presence of viral RNA using RT-qPCR.\u003c/p\u003e\u003ch2\u003eSialidase inhibition assay\u003c/h2\u003e\u003cp\u003eThe heat-inactivated immune sera of all animals in a group were mixed to obtain group-specific serum pools for both the primary and the booster vaccinations. Starting with a dilution of 1:20, four-fold serially diluted serum pools were prepared in 96-well plates using PBS as diluent. To 50 µL of each serum dilution, 50 µL of NA reassortant H5N1 virus (10\u003csup\u003e7\u003c/sup\u003e ffu/mL) were added (in duplicates) and incubated for 30 min at 37°C, The serum-virus mixture (100 µL) was then transferred to Nunc MaxiSorp 96-well plates (Thermo Fisher Scientific) that had been coated for 24 h at 4°C with 10 µg/well of bovine fetuin (Merck KGaA, Darmstadt, Germany, cat. no. F2379) and subsequently blocked with 1% bovine serum albumin (BSA, Fluka Chemie GmbH, Buchs, Switzerland, cat. no. 05480). The microtiter plates with the immobilized fetuin were incubated with the serum-virus mixtures for 20 h at 37°C and then washed three times with 250 µL/well of PBS containing 0.05% Tween 20 (PBS-T). Subsequently, 50 µL/well of PBS containing 0.1% BSA and 0.5 µg/mL of biotinylated peanut agglutinin (PNA, Merck KGaA cat. no. L6135) were added and incubated for 1 h at 21°C. The microtiter plates were washed three times with PBS-T before 50 µL/well of streptavidin-peroxidase conjugate (Dako, Glostrup, Denmark; 1:5000 in PBS) were added and incubated with the plates for 30 min at 21°C. The wells were washed as above again and 50 µL of 3,3′,5,5′-Tetramethylbenzidine (TMB) peroxidase substrate (Merck KGaA, cat. no. T4444) was added to each well. The reaction was stopped by adding 50 µL/well of 1 M hydrochloric acid (Merck KGaA, cat. no. 1.09057.1000) and absorption was measured at 450 nm using the GloMax® Discover microplate reader (Promega Corporation, Madison, WI, USA). The OD\u003csub\u003e450\u003c/sub\u003e values were plotted against the serum dilution factors, and the dilution factor resulting in 50% inhibition (inhibitory concentration 50% [IC\u003csub\u003e50\u003c/sub\u003e]) was calculated by non-linear regression analysis (curve fitting).\u003c/p\u003e\u003ch2\u003eVirus neutralization test\u003c/h2\u003e\u003cp\u003ePorcine sera were heated for 30 min at 56°C to inactivate complement factors. Serial two-fold dilutions in MEM medium were added in quadruplicates to 96-well microtiter plates (50 µL/well). To each well, 50 µL of H1N1\u003csub\u003eHH4/09\u003c/sub\u003e, H1N1\u003csub\u003eVic/19\u003c/sub\u003e or H5N1\u003csub\u003eTex/24\u003c/sub\u003e (2000 ffu/mL) were added and incubated for 1 h at 37°C. The antibody/virus mix was incubated for 1 h at 37°C and 5% CO\u003csub\u003e2\u003c/sub\u003e with MDCK-II cells that were grown to confluence in 96-well cell culture plates. Thereafter, the cells were washed once and incubated for 24 h at 37°C with 100 µL/well of MEM medium. The cells were fixed with 4% formalin and permeabilized with PBS containing 0.25% Triton X-100. Virus-infected cells were visualized by indirect immunofluorescence using a monoclonal antibody directed to the influenza nucleoprotein (NP) antigen (American Type Culture Collection, Manassas, Virginia, USA, ATCC HB-65, clone H16-L10-4R5, diluted 1:50 with PBS) and goat anti-mouse IgG conjugated with Alexa Fluor-488 (Life Technologies, Zug, Switzerland; diluted 1:500 in PBS) \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. The 50% neutralizing dose (ND\u003csub\u003e50\u003c/sub\u003e) was calculated using the Spearman-Kärber method \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003ch2\u003eVirus release inhibition assay\u003c/h2\u003e\u003cp\u003eMDCK cells were grown in 96-well tissue culture plates and inoculated for 30 min at 37°C with 50 µl MEM containing 100 ffu of H5N1\u003csub\u003eTex/24\u003c/sub\u003e. The cells were washed once with 200 µL of Dulbecco`s phosphate-buffered saline (DPBS; Life Technologies, cat. no. 14040-091) and incubated at 37°C with 200 uL of MEM cell culture medium containing serially two-fold diluted immune sera (boost) and supplemented with penicillin and streptomycin (Life Technologies, cat. no. 15140-122). At 24 h and 48 h post infection, 100 µL aliquots of cell culture supernatant were collected and frozen at -70°C. The number of infectious virus particles released was determined as described above (see section “Titration of influenza A viruses”).\u003c/p\u003e\u003ch2\u003eRT-qPCR\u003c/h2\u003e\u003cp\u003eSwab samples were directly transferred to 700 µl of RA1 lysis buffer (Macherey-Nagel, Düren, Germany, cat. no. 740961) containing 1% β-mercaptoethanol. Organs were homogenized in RA1 lysis buffer using a tissue bullet blender (Next Advanced Inc., Troy, NY, USA). Total RNA was extracted from the lysates using the NucleoMag Vet kit (Macherey-Nagel, cat.no. 744200) according to the manufacturer’s protocol. Reverse transcription from RNA to cDNA and real-time quantitative PCR (qPCR) were performed on a QuantStudio 5 real-time PCR system (Thermo Fisher Scientific) using the AgPath-ID One-Step RT-PCR kit (Life Technologies, Zug, Switzerland, cat. no. AM1005) with vRNA segment 7-specific oligonucleotide primers and probe \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Data were acquired and analyzed using the Design and Analysis Software v1.5.2 (Thermo Fisher Scientific).\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using GraphPad Prism 10, version 10.1.2 (GraphPad Software, Boston, Massachusetts, USA). Unless noted otherwise, data are presented as scatter dot plots with geometric mean values and 95% confidence interval (CI) also indicated. Specific statistical tests such as the one-way or two-way ANOVA test were used to assess significant differences in serum antibody responses in vaccinated animals as indicated in the figure legends. \u003cem\u003eP\u003c/em\u003e values \u0026lt; 0.05 were considered significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe like to thank the Swiss National Research Foundation (SNSF) and the European Union\u0026rsquo;s Horizon Europe Project 101136346 EUPAHW for financial support of this project. We are grateful to Daniel Brechb\u0026uuml;hl and Katarzyna Sliz for their support in animal experimentation. We like to thank Martin Schwemmle (University of Freiburg, Germany) and Yoshihiro Sakoda (University of Sapporo, Japan) for providing plasmids and Diego Diel (Cornell University, USA) for providing the bovine H5N1 virus isolate. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAS and GZ conceived the study and were responsible for funding acquisition; NR, OG and GZ conceptualized and conducted the animal experiments; RA, LB, and GZ generated recombinant influenza viruses and VSV replicon vaccines; LB performed RT-qPCR analysis, virus neutralization tests, virus release inhibition tests, and sialidase inhibition tests; LB and GZ analyzed and evaluated the data. GZ wrote the manuscript draft.\u0026nbsp;All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files. Raw data are available at Zenodo (\u003cstrong\u003ehttps://doi.org/10.5281/zenodo.17302034\u003c/strong\u003e\u003cstrong\u003e).\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRA, GZ, and AS filed a patent application related to the intramuscular prime/intranasal boost vaccine described in this work. All other authors declare no competing interests. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work has received funding from the Swiss National Research Foundation (SNSF), grant \u0026nbsp;no. 189903 (A.S., G.Z.; https://data.snf.ch/grants/grant/189903) and was co-funded by the European Union\u0026rsquo;s Horizon Europe Project 101136346 EUPAHW by a grant to A.S.. The funders had no role in study design, data collection and analysis, decisions to publish or preparation of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWu, N. C. \u0026amp; Wilson, I. A. A Perspective on the Structural and Functional Constraints for Immune Evasion: Insights from Influenza Virus. \u003cem\u003eJ Mol Biol\u003c/em\u003e \u003cstrong\u003e429\u003c/strong\u003e, 2694-2709, doi:10.1016/j.jmb.2017.06.015 (2017).\u003c/li\u003e\n\u003cli\u003eWu, N. C. \u0026amp; Wilson, I. A. 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A., Renzullo, S. \u0026amp; Baumer, A. Phylogenetic characterization of H5N1 highly pathogenic avian influenza viruses isolated in Switzerland in 2006. \u003cem\u003eVirus Genes\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 407-413, doi:10.1007/s11262-008-0285-2 (2008).\u003c/li\u003e\n\u003cli\u003eSpackman, E.\u003cem\u003e et al.\u003c/em\u003e Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. \u003cem\u003eJ Clin Microbiol\u003c/em\u003e\u003cstrong\u003e40\u003c/strong\u003e, 3256-3260, doi:10.1128/JCM.40.9.3256-3260.2002 (2002).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Neuraminidase, hemagglutinin, RNA replicon particle, virus shedding, 2009 pandemic H1N1 influenza virus, H5N1 highly pathogenic avian influenza virus, prime-boost vaccination ","lastPublishedDoi":"10.21203/rs.3.rs-7818522/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7818522/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSeasonal influenza viruses escape the human immune response by antigenic drift, i.e. the positive selection of point mutations that prevent the binding of inhibitory antibodies to the influenza antigens HA and NA. The efficacy of seasonal influenza vaccines can be less than 50% if the selected influenza vaccine strain does not match the antigenic characteristics of the circulating seasonal influenza virus. In this study, we used the porcine model to evaluate the efficacy of an RNA replicon vaccine encoding the HA and NA antigens of A/Hamburg/4/2009 (H1N1) (H1N1\u003csub\u003eHH4/09\u003c/sub\u003e) in inducing cross-reactive immunity. We found that a single intramuscular immunization with this vaccine elicited high levels of antibodies with H1N1\u003csub\u003eHH4/09\u003c/sub\u003e-neutralizing activity and potent N1-sialidase inhibition. A second immunization with the same H1/N1 RNA replicon particles or with a live-attenuated influenza vaccine (LAIV) based on a modified H1N1\u003csub\u003eHH4/09\u003c/sub\u003e virus boosted the inhibitory activity of the immune sera against the antigen-drifted A/Victoria/2570/2019 (H1N1) (H1N1\u003csub\u003eVic/19\u003c/sub\u003e) strain. Interestingly, vaccination elicited N1-specific antibodies that also inhibited the activity of avian N1 sialidase and potently inhibited the replication of A/cattle/Texas/063224-24-1/2024 (H5N1) (H5N1\u003csub\u003eTex/24\u003c/sub\u003e) \u003cem\u003ein vitro\u003c/em\u003e. When challenged nasally with a H1N1\u003csub\u003eHH4/09\u003c/sub\u003e /H1N1\u003csub\u003eVic/19\u003c/sub\u003e 6:2 reassortant virus encoding the HA and NA antigens of H1N1\u003csub\u003eVic/19\u003c/sub\u003e, immunized pigs did not shed infectious virus while the control animals did, suggesting that homologous prime/boost vaccination with H1/N1 replicon particles can block virus replication in the upper respiratory tract as efficiently as the heterologous RNA replicon prime/LAIV boost immunization regimen. In conclusion, RNA replicons encoding both HA and NA either used alone or in combination with LAIV mediate protection against antigen-drifted influenza viruses and reduce the risk of vaccination breakthroughs due to antigen mismatch. Furthermore, this vaccine may also limit the infection by zoonotic H5N1 viruses.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"An RNA replicon vaccine encoding HA and NA prevents shedding of antigen- drifted 2009 pandemic H1N1 influenza virus in the pig model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-06 18:26:49","doi":"10.21203/rs.3.rs-7818522/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-28T06:16:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-26T18:07:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"63804072566873410200948968260110947431","date":"2026-01-12T17:25:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-19T22:27:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"14771255808714696084013421455415906877","date":"2025-12-02T23:21:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"87574456105913059029841610328537141261","date":"2025-10-28T08:41:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-27T20:49:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-27T20:47:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-24T03:33:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Vaccines","date":"2025-10-09T14:19:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"254c397c-963c-4fa5-a695-e8207e258d59","owner":[],"postedDate":"November 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":57446005,"name":"Biological sciences/Immunology"},{"id":57446006,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-03-30T16:24:20+00:00","versionOfRecord":{"articleIdentity":"rs-7818522","link":"https://doi.org/10.1038/s41541-026-01428-6","journal":{"identity":"npj-vaccines","isVorOnly":false,"title":"npj Vaccines"},"publishedOn":"2026-03-28 16:11:54","publishedOnDateReadable":"March 28th, 2026"},"versionCreatedAt":"2025-11-06 18:26:49","video":"","vorDoi":"10.1038/s41541-026-01428-6","vorDoiUrl":"https://doi.org/10.1038/s41541-026-01428-6","workflowStages":[]},"version":"v1","identity":"rs-7818522","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7818522","identity":"rs-7818522","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}