An experimental universal swine influenza a virus (IAV) vaccine candidate based on the M2 ectodomain (M2e) peptide does not provide protection against H1N1 IAV challenge in pigs


Swine flu is a common disease problem in North American pig populations and swine influenza A viruses (IAV) are extremely diverse and the lack of cross protection between heterologous strains is impacting vaccine efficacy in the field. The objective of this study was to design and test a novel swine flu vaccine targeting the M2 ectodomain (M2e) of IAV, a highly conserved region within the IAV proteome. In brief, an M2e peptide was designed to match the predominant swine IAV M2 sequence based on global analysis of sequences from pigs and humans. The resulting sequence was used to synthesize the M2e peptide coupled to a carrier protein. The final vaccine concentration was 200 µg per dose, and a commercial, microemulsion-based aqueous adjuvant was added. Nine 3-week-old IAV negative piglets were randomly assigned to three groups and rooms including non-vaccinated pigs (NEG-CONTROLs) and vaccinated pigs using the intramuscular (M2e-IM) or the intranasal route (M2e-IN). Vaccinations were done at weaning and again at 2 weeks later. An in-house enzyme-linked immunosorbent assay (ELISA) was developed and validated to study the M2e IgG antibody response and demonstrated M2e-IM pigs had a higher systemic antibody response compared to M2e-IN pigs. Subsequently, an IAV challenge study was conducted. The results indicated that M2e-IM vaccinated pigs were not protected from H1N1 (US pandemic clade, global clade 1A.3.3.2) challenge despite having a strong humoral anti-M2e immune response. In conclusion, while the experimental IAV vaccine was able to induce anti-M2e antibodies, when challenged with H1N1, the vaccinated pigs were not protected, perhaps indicating that reactivity to the M2e antigen alone is not sufficient to reduce clinical signs, lesions or shedding associated with experimental IAV challenge.

    1. Introduction

    Influenza A viruses (IAV) in North American pigs are genetically diverse and include H3N2, H1N1 and H1N2 subtypes [1], [2]. Similar trends are also being reported from Europe [3]. Notably, there is a lack of cross protection between dissimilar or heterologous strains [4]. IAV is an enveloped, negative sense, single stranded, segmented RNA virus that belongs to the family Orthomyxoviridae [5]. The two major glycoproteins of IAV are hemagglutinin (HA) and neuraminidase (NA) that are responsible for viral entry and viral release from infected cells [6], [7]. These two glycoproteins are the main targets for the humoral immune response [8], [9], [10].

    In pigs, IAV is endemic in many herds and causes respiratory disease of varying severity from subclinical to severe acute outbreaks characterized by persistent coughing, labored respiration, anorexia and lethargy with high morbidity but low mortality [11]. Among pigs, IAV can be transmitted through direct contact, fomites, and aerosols [12]. Some pigs may have no clinical signs (subclinical infection) although still shed and transmit the virus [13]. The IAV infected epithelial cells of bronchi, bronchioles and alveoli can become severely compromised due to inflammatory cell influx, which leads to cell necrosis, and obstruction of the airways [12].

    Pigs are important in the transmission of IAV to other hosts due to their propensity to be a mixing vessel for human and avian viruses due to having similar receptors as people and birds [14]. Transmission of pig-origin IAV to people with close contact, especially at county fairs in the USA, has been reported [15], [16].

    Vaccination of pigs against IAV in the U.S. is commonly performed in breeding herds to reduce clinical disease in the dams and abortions [17]. Breeding herd vaccination also provides offspring with passively derived antibodies which may protect piglets until weaning [18]. There are several licensed, commercial IAV vaccines available in the U.S. [11]. They are all based on inactivated virus material, subunit vaccines or alpha replicon virus based, and in general provide some level of protection against similar or homologous IAV [11]. The use of a highly conserved IAV peptide as the basis for a vaccine for pigs may be an approach to better protect against multiple strains of IAV that may circulate within a population of pigs over time [19]. The introduction of new strains can occur through contact with incoming IAV positive pigs, contact with wild birds, or contact with infected humans [20].

    Both the norovirus P particle vaccine platform, derived from the protruding (P) domain of the norovirus VP1 capsid protein [21], [22], and large complex-based vaccines containing the M2e epitope have been produced in E. coli with high yield and stability [23], [24]. In mouse experiments, both vaccines proved to be highly immunogenic, and provided full protection against lethal IAV challenge [23], [24]. However, tests in pigs with these vaccines were not successful [25]. P-particle and complex based vaccines are difficult to produce at high concentrations and production is also expensive precluding further pursuit of these vectors. The M2e protein is found within the viral envelope of IAV, and it aids in viral uncoating during viral invasion of cells [26]. The M2e epitope is found on the surface of IAV infected cells, and due to the highly conserved nature of the epitope, it has been suggested as the perfect target for a universal IAV vaccine [27], [28]. However, investigations by our group indicated species specific differences among M2e sequences suggesting that a universal M2e sequence requires a selection process.

    The objective of this study was to design and produce a novel inexpensive M2e peptide vaccine for use in pigs, test the immunogenicity of the vaccine, and conduct a vaccine challenge study to assess efficacy.

    2. Methods

    2.1. Ethical statement.

    The Iowa State University Institutional Animal Care and Use Committee approved this study (Approval number: IACUC 18-191). The efforts to alleviate suffering in this study included defined endpoints for each pig and independent veterinary supervision for the duration of the project. Various types of environmental enrichment were provided and the pigs were regularly checked by veterinarians unrelated to project personnel.

    2.2. M2e peptide selection

    Our objective was to design a peptide corresponding to the M2 ectodomain that represented as broad of a spectrum of swine influenza viruses as possible. To identify the diversity of recent IAV M2e sequences, the Influenza Research Database (

    ), was searched on the 15th and 16th of January 2019 for full length M2 protein sequences. Search criteria and numbers of sequences returned are indicated in Table 1. Sequences were trimmed to the ectodomain and aligned using Muscle [29]. The alignments were then exported as fasta files (manually adding the fa suffix) and logos computed using WebLogo (

    ), as a means of graphically identifying both variability and a consensus sequence. To identify a consensus for the genotype 1 H3N2v swine virus causing zoonotic infections in the USA [29], [30], the following accession codes (JX092635, JQ738174, JQ738182, JX534985, JX534993, JX535001, JQ350525, JX092395, JX092396, JX092398, JX092414, JX092611, JX092612, JX092409, JQ738158, JQ738166, JQ738150, JX092623, JN940425, JF812322, JF812323, JQ689135 and JX280453) were manually downloaded and similarly processed.

    Table 1. Search criteria and numbers of M2 protein sequences returned from the IRD on January 15th-16th 2019. Reduced date ranges were used for the human viruses to keep the number of returned sequences within the capacity of the Weblogo software.

    Host Region Subtype Earliest sample date Number
    Swine North America all 2014 3208
    Swine North America H1N1 2014 979
    Swine North America H1N2 2014 1114
    Swine North America H3N2 2014 1009
    Swine European all 2014 75
    Swine Asia all 2009 705
    Human Global H1N1 2016 2852
    Human Global H3N2 2017 3222

    2.3. Experimental design

    Initially, the M2e vaccine was designed, produced and an appropriate adjuvant was selected. This was followed by a small pilot immunization study (Fig. 1) in which the experimental M2e antibody response was evaluated by enzyme-linked immunosorbent assay (ELISA) and western blots and to develop an M2e serologic assay for future use. In brief, nine 3-week-old pigs were randomly assigned to a single room and three experimental groups: unvaccinated negative control group (NEG-CONTROL), a group vaccinated twice intramuscularly with the M2e vaccine (M2e-IM), and a group vaccinated twice with the M2e vaccine using the intranasal route (M2e-IN). Subsequently, a vaccine-challenge study was conducted to assess the vaccine efficacy against a 2017 North American pig H1N1 strain (US pandemic clade, global clade 1A.3.3.2). The challenge study consisted of 30 3-week old pigs purchased from the same farm as the pilot study and were randomly assigned to 3 rooms and groups with 10 pigs each. This included a NEG-CONTROL group (no vaccination, no challenge), a POS-CONTROL group (no vaccination, IAV challenge) and an M2e-IM-FLU group (intramuscular vaccination, IAV challenge). Blood was collected weekly from the pigs in both studies to obtain serum for anti-IAV-antibody assessment by ELISA. Pigs were observed daily for 3 days for evidence of any vaccine reaction and pigs were clinically assessed every day after challenge in addition to daily nasal swab collections for assessment of IAV shedding.

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    Fig. 1. Experimental design. The immunization study is on top of the timeline and the vaccine-challenge study is below the timeline. Abbreviations: NS, nasal swab collection for the vaccine -challenge study only; dpv, day post vaccination; dpc, day post challenge; S, serum collection for both study parts except for dpv 31 and 33 where serum was only collected from pigs during necropsy.

    2.4. Peptide source and vaccine formulation

    The M2e vaccine peptide as described in the previous section was commercially synthesized (Thermo Fisher Scientific, USA). Due to its small size, the peptide was conjugated to the carrier protein keyhole limpet hemocyanin (KLH) (Thermo Fisher Scientific). Shortly before vaccination, the peptide was diluted in PBS (pH 7.4) to a final concentration of 200 µg per dose, which was based on available data from the literature on similar IAV vaccination trials [31], [32]. The diluted peptide was then combined with the commercial, micro-emulsion-based aqueous adjuvant Montanide™ IMS 1313 VG N ST (SEPPIC, VA, USA). This adjuvant is compatible with phosphate buffered solutions (PBS) and expected to induce a strong and rapid immune response as well as sustainable protection for a two-dose protocol and can be used by parenteral or via mucosal routes [33].

    2.5. Animal source and housing and feed

    Thirty-nine 3-week-old, mixed-gender crossbred pigs, were purchased from a commercial high health breeding herd without evidence of IAV circulation based on regular serological testing on blood and PCR testing on nasal swabs. The pigs came in two batches of 9 and 30 pigs approximately 6 months apart to allow time for testing the vaccine formulation and ELISA development. The pigs were transported to research facilities at the College of Veterinary Medicine, Iowa State University, Ames, Iowa, US. The study was conducted under biosafety level 2 (BSL-2) conditions. For the immunization study, pigs were housed in a single room with 9 pigs in a pen and for the challenge study in three separate rooms with 10 pigs per room and pen. Each room pen was approximately 30 m2 and equipped with a nipple waterer and feed was provided ad libitum. The pigs were fed an age-appropriate ration free of antibiotics and animal proteins (Heartland Co-Op, Prairie city, IA, USA).

    2.6. Vaccination

    Each pig received 2 mL of the vaccine containing 200 µg of peptide by the intranasal (IN) or the intramuscular (IM) route, according to the assigned group. For the IN route, a mucosal administration device (MAD; Teleflex Medical, Morrisville, NC, USA) was used. The MAD allows for fine dispersion of the vaccine resulting in better distribution within the nasal cavity as described [34]. For both studies, vaccination was performed after weaning (between 3 and 4 weeks of age). In brief, the IM administration was done in the right site of the neck by using a 3 mL syringe and a 22 gauge × 1.5-inch needle. A person restrained the pig by holding it while another person administered the vaccine. For the second vaccination, the left site of the neck was injected. For the IN administration, the MAD device was connected with a 3 mL syringe. A handler held the pig upright and the vaccine was sprayed into as a fine mist alternating into each nostril. Each pig was vaccinated using a new needle. The vaccine/challenge study used only the IM administration route as described above. All vaccinated pigs received a booster vaccination 14 days after the initial immunization. Negative control pigs remained unvaccinated.

    2.7. Sample collection

    Blood samples were collected from each pig in serum separator tubes (Fisher Scientific, Pittsburgh, Pennsylvania, USA) weekly at −5, 7, 14, 21, 28, and 31 days post vaccination (dpv) (immunization study) or 33 dpv (vaccine-challenge study only). Once collected, the serum separator tubes were centrifuged at 1500g for 8 min at 4 °C, and serum aliquoted and stored at −80 °C until testing. For the vaccine-challenge study, peripheral blood mononuclear cells (PBMCs) were collected at the time of IAV challenge corresponding to dpv 28 to conduct an enzyme-linked immunosorbent spot (ELISpot) assay. In brief, 8–10 mL blood was collected from each pig using BD Vacutainer® CPT™ cell preparation tubes with sodium citrate (Becton, Dickinson and Company) and processed within 2 h of collection. Cotton-tipped swabs (Fisher Scientific, Pittsburgh, PA, USA) were used to collect nasal secretions by swabbing both nostrils at day post challenge (dpc) − 1, 1, 2, 3, 4 and 5. After collection, the swabs were immediately placed in 1 mL of phosphate buffered saline (PBS) in 5 mL plastic tubes and stored at − 80 °C until testing.

    2.8. Clinical assessment

    All pigs were weighed upon arrival, at challenge (if applicable) and at necropsy. Rectal temperature, injection site reactions, cough and respiratory scores were assessed the first three days following the first vaccination (immunization study, vaccine-challenge study), and at challenge and dpc 1, 2, 3, 4 and 5 (vaccine-challenge study). The scoring system for cough ranged from 0 = absent, 1 = single cough, 2 = persisting cough, and the respiratory scores ranged from 0 = normal to 6 = severe dyspnea and/or tachypnea when at rest [35]. Pigs with rectal temperatures equal to or greater than 40.5 °C were considered febrile.

    2.9. M2e enzyme-linked immunosorbent assay (ELISA) development and optimization

    Initially, the vaccine peptide was used (i.e. M2e bound to KLH, M2e-KLH) to coat the ELISA plates. As KLH was also in the vaccine, antibodies against KLH prevented the ability to differentiate antibodies specific to M2e. An avian influenza A M2e synthetic peptide (Thermo-Fisher, IL, USA, Cat. #: PEP-0533; M23-avian-commercial) was used to detect M2e-specific antibody responses. The peptide sequence was proprietary; however, the manufacturer indicated the peptide corresponds to 15 amino acids near the amino terminus of H5N1 influenza A M2. Ultimately, the selected peptide was bound to BSA as protein (M2e-BSA) avoiding any protein cross reactivity with the vaccine peptide bound to KLH. For the ELISA development, for each of the three M2e peptides (M2e-BSA, M2e-KLH and M2e-avian-commerical), four standardization plates were tested in three rounds to determine the best serum sample dilution, secondary antibody dilution, and plate composition for the in-house M2e ELISA. Initially an ELISA plate (96-well flat-bottom plate, MaxiSorp, Nunc, Roskilde, Denmark) was coated with phosphate-buffered saline pH 7.4 (PBS) and carbonate-bicarbonate pH 7.4, and tested using serum dilutions of 1:20, 1:50, and 1:100. The following samples were used for plate validation: serum samples from the immunization study (three samples from the IM-M2e group, three samples from the IN-M2e group, and two samples from the NEG-CONTROLS collected on 21 dpv and serum samples from a previous project [36]including two samples from IAV negative control pigs, and two samples from vaccinated and IAV challenged pigs [40 d after vaccination with a commercial IAV vaccine (FLUSURE XP®, Zoetis Inc., Kalamazoo, Michigan, USA) and 4 days after challenge with a pH1N1 2017 isolate]. An IAV M2e concentration of 2 µg/mL diluted in PBS was found to be ideal, and a second standardization test plate was coated using these conditions. Serum tested on the second validation plate were diluted 1:50 and 1:100. The same three IN-M2e serum samples, and three IM-M2e serum samples were tested on this plate along with three IAV negative samples from the current study. The positive and negative controls used for a third test were from different pigs from the previous study to provide a wider range of comparison values.

    2.10. Final M2e ELISA conditions

    The final ELISA conditions using the M2e-BSA conjugated peptide were 100 µL of antigen solution per well with serum samples diluted at 1:100 concentration in sera diluent PBS blocking buffer. Specifically, the ELISA plate was coated with 100 µL/well of antigen solution and incubated overnight at 4 °C after having been sealed. The antigen solution was removed, and the plate was washed 5 times with wash buffer (PBS 1x pH 7.4 + 0.05 % Tween-20, Mediatech Inc., VI, USA). Next, 300 µL of blocking buffer (Pierce Protein-free T20 PBS Blocking Buffer, Thermo-Fisher) was added to each well, and the plate was incubated for one hour at room temperature. The blocking buffer was removed, and the plates were dried, without any additional wash step, at 37 °C for 2–4 h and stored at 4 °C until use. The serum samples were diluted at 1:100 with a serum diluent (Pierce Protein-free T20 PBS Blocking Buffer, Thermo-Fisher), and 100 µL of diluted serum samples were added to each well and incubated at 37 °C for 45 min. The plate was then washed five times with washing buffer. The secondary antibody [Goat anti-swine IgG (H + L) HRP-conjugated, Jackson ImmunoResearch Lab. Inc., PA, USA] was diluted 1:5,000 with serum diluent. A total of 100 µL of diluted conjugate was added to each well, the plate was incubated at 37 °C for 30 min, and then washed five times with wash buffer. The third incubation step began with 100 µL of Sureblue TMB Microwell Peroxidase substrate (KPL, Gaithesburg, ML, USA) added to each well, and the plate was incubated for 15 min at room temperature in the dark. The reaction was stopped with 100 µL of stop solution (KPL TMB Stop Solution, Seracare, Milford, MA, USA) added per well and read at 450 nm.

    2.11. Commercial IAV nucleoprotein ELISA

    A commercial nucleoprotein (NP) blocking ELISA (Swine Influenza Virus Ab Test, IDEXX, WI, USA,) was used to determine the presence of anti-NP IAV in the serum samples. This ELISA considers a sample positive if the sample-to-negative (S/N) ratio is <0.6, and negative if the S/N was >0.6. All of the serum samples collected at dpv −5 and 28 and also on dpc 5 (vaccine/challenge study only) were tested using this ELISA. The assay was conducted at the Veterinary Diagnostic Laboratory at Iowa State University.

    2.12. Western blot

    Madin-Darby Canine Kidney (MDCK)- 2,6-sialtransferase (SIAT cells), derived by the stable transfection of MDCK cells with the cDNA of human SIAT1, were infected with IAV H1N1 strain A/Puerto Rico/8/1934 (NCBI:txid211044) or A/California/04/2009 (NCBI:txid641501) at a multiplicity of infection (MOI) of 5 for 16 h. Cells were then lysed with Laemmli sample buffer. Samples were separated under reducing conditions using SDS-PAGE (Bio-Rad Laboratories, CA, USA) and proteins were transferred to nitrocellulose membrane (Bio-Rad Laboratories). The membrane was blocked with Intercept blocking buffer (LI-COR Biosciences, NE, USA) for 1 h and incubated with 1:20 or 1:50 diluted pig sera and 1:5000 diluted anti-beta actin (Abcam plc, England, UK) overnight at 4 °C. The membrane was then incubated with fluorescent secondary antibodies (LI-COR Biosciences) for 1 h at room temperature and detection was conducted using Odyssey Fc Imager (LI-COR Biosciences, USA).

    2.13. Enzyme-linked immunospot (ELISpot) assay

    Within 2 h of blood collection, the tubes were centrifuged at 1800g for 20 min at room temperature. The buffy coat was collected and resuspended in PBS. Cells were washed and centrifuged at 500g for 5 min at 4 °C, the supernatant was discarded, and the pellet was used immediately for the ELISpot assay. A previously described commercial IFNγ ELISpot kit (Porcine IFN-gamma ELISpot kit, R&D Systems Inc, Minneapolis, MN, USA) was used per the manufacturer’s directions with 50 µL of complete RPMI added to each well [36]. A total of 2.5 x 105 viable PBMC in 100 mL of complete RPMI 1640 media supplemented with 10 % heat-inactivated fetal bovine serum were seeded into pretreated microplates. The seeded cells were stimulated with the challenge pH1N1, A/Swine/Iowa/A01104104/2017(H1N1) and an H3N2 strain, A/Swine/Iowa/A02135000/2017(H3N2), both at a concentration titer of 5.8 log10 median tissue culture infectious dose (TCID50) per mL. For control purposes, PBMCs were stimulated with 0.25 µg pokeweed mitogen (MP Biomedicals™, Santa Ana, CA, USA) in 100 µL of complete RPMI. The cells were then incubated for 36 h at 37 °C in a 5 % CO2 incubator. Subsequently, the ELISpot assay was performed according to the manufacturer’s instructions. Blue-black colored precipitate spots corresponding to activated IFNγ secreting cells were counted with an ELISpot reader (ImmunoSpot ELISpot analyzer, Cellular Technology Limited, Cleveland, OH, USA).

    2.14. IAV PCR

    Nucleic acids were extracted from nasal swabs using the MagMAXTM Pathogen RNA/DNA kit (Thermo Fisher Scientific) and a Kingfisher Flex instrument (Thermo Fisher Scientific) following the manufacturer’s instructions. For each sample, 100 μL was used for extraction, and nucleic acids were eluted into 90 μL of elution buffer as described [37]. A quantitative real-time reverse transcription (RT) PCR assay was performed using a VetMAX™-Gold SIV Detection Kit (Applied Biosystems, Life Technologies, Foster City, CA, USA) per manufacturer’s instructions and based on a standard curve using 50 % tissue culture infectious dose per ml of an IAV isolate. A sample with a cycle threshold (ct) value below 38 cycles was considered positive. Suspect samples with a ct between 38 and 40 cycles were considered negative. Appropriate negative and positive controls were included in each run.

    2.15. IAV challenge

    For the IAV inoculation in the vaccine/challenge study, a pH1N1 isolate (A/swine/Iowa/A01104104/2017) was used at a dose of 105 TCID50 per ml. Each pig received 2 mL of the inoculum intranasally and 2 mL intratracheally. For the challenge, the pigs were anaesthetized using a ketamine (8 mg/kg), xylazine (4 mg/kg), and telazol (6 mg/kg) combination as described [38]. NEG-CONTROL pigs were mock challenged with medium alone.

    2.16. Necropsy and macroscopic and microscopic lesion assessment

    On dpc 5, all pigs were euthanized by intravenous administration of pentobarbital overdose (FATAL-PLUS®, Vortech Pharmaceuticals LTD, Dearborn, MI, USA). A pathologist (PCG) blinded to the pig treatment status assessed the lung lesions based on the percentage of lung surface affected [39]. Three fresh lung sections from the right cranial lobe, the right middle lung lobe and the accessary lung lobe and one section of distal trachea were collected in 10 % neutral-buffered formalin and processed for histopathology. Microscopic lung lesions were assessed by a veterinary pathologist (PCG) blinded to the pig treatment status [39]. Specifically, the percentage of intrapulmonary airway epithelial necrosis and magnitude of peribronchiolar lymphohistiocytic cuffing were scored ranging from 0 = no lesion to 4 = severe diffuse lesions. Immunohistochemistry (IHC) was used to assess the intralesional amount of IAV antigen as described [40], [41] in lung epithelium, lung parenchyma, and trachea with scores ranging from (0) none, (1) few cells with positive labeling, (2) mild scattered labeling, (3) moderate scattered labeling, (4) abundant scattered labeling (greater than 50 %epithelium positive in affected airways).

    2.17. Statistical analysis

    Summary statistics were calculated for groups to assess the distributional property. Quantitative RT-PCR data was log transformed prior to analysis. Repeated measures (nasal shedding and rectal temperature) were analyzed by ANOVA using Tukey’s honest significant difference. The null hypothesis rejection level was P < 0.05. Non-repeated measures were assessed using nonparametric Kruskal-Wallis ANOVA. When group variances were different, pair-wise comparisons were performed using the Wilcoxon rank sum test. Differences in incidence were evaluated using Fisher’s exact test. Correlations were estimated by Pearson’s method. All analyses were performed with JMP® Pro Version 16.2.0 statistical software.

    3. Results

    3.1. M2e peptide design

    Our objective was to design a peptide corresponding to the IAV M2 ectodomain that represented as broad a spectrum of swine influenza viruses as possible. To identify the diversity of recent swine-derived M2e sequences whilst considering geographic and viral antigenic diversity, the Influenza Research Database (

    ), was searched for full length M2 protein sequences filtered by date range, region of virus isolation and viral subtype (Table 1). For comparison, date-limited searches were also performed for recent human H1N1 and H3N2 seasonal IAV isolates. A small sample (23) of human genotype 1 H3N2v M2 sequences [30] were also included in the analysis to consider an important swine-derived source of zoonotic infection. Sequences were aligned and consensus sequences determined. This indicated that the majority of swine IAV isolates from North America and Asia had an M2e ectodomain that was identical to that of the swine-derived human H1N1 2009 pandemic (pdm09) virus (Fig. 2). The consensus sequence of the North American swine M2e sequences was not affected by the antigenic subtype of virus (data not shown). The pdm09 M2e consensus also extended to the zoonotic H3N2v viruses, as expected from their origin [30]. However, also as expected from its evolutionary lineage [42], the M2e consensus of human seasonal H3N2 viruses diverged, as did the M2e sequence of European swine isolates. Considering the numbers of available sequences for North American swine IAVs and their likely antigenic similarity with human pdm09 viruses, the immunizing peptide was designed to be a match for the pdm09 ectodomain, modified to avoid problematic cysteine chemistry (Fig. 2).

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    Fig. 2. Consensus sequences of M2e domains from swine and human IAVs circa 2014–2018. Amino acids in red indicate deviations from the 2009 H1N1 swine-derived human pandemic consensus. Underlined characters indicate sequence changes made in the synthetic peptide to avoid problems with cysteine chemistry. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    3.2. Vaccine reactions, immunization study serology results and selection of the vaccination route

    Both vaccination routes worked well and vaccine reactions were not observed post-vaccination (0–3 dpv). At arrival, all pigs were tested for antibodies to IAV using the commercial assay based on the IAV NP and the experimental M2e ELISA with the M2e-BSA coating. Serum samples from all pigs were negative at arrival. Seroconversion was first seen at dpv 14 in both the M2e-IN and the M2e-IM groups. The M2e-IM pigs produced the highest antibody titers experiment (Fig. 3). Based on the IM route inducing the highest seroconversion rates, the IM route was utilized in the vaccine-challenge study.

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    Fig. 3. Seroconversion in pigs vaccinated with M2e via the intranasal (IN) or the intramuscular (IM) route. Different superscripts (a,b) at a given day post vaccination (dpv) indicate significant differences among groups. The green dotted line is the M2e ELISA cut-off, an S/P ratio of 0.5. Abbreviations: S/P, sample to positive ratio. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    3.3. Western blot

    To confirm M2e antibodies were not detected with the M2e ELISA, and hence potential interference with vaccination, a western blot was done using selected serum samples from the immunization study. Target cell lysates from MDCK-SIAT cells were prepared that had been infected with either Cal04 (pdm09) or PR8 (old human H1N1), or mock infected. When the lysates were validated it appeared there was even loading. The positive control PR8-infected sample had two obvious new polypeptide species which appeared to have the right size for NP (58 kDa) and M1 (∼25 kDa). The other control, Cal04 lysate, had lower amounts of presumed NP and M1, which was expected, as even in the SIAT cell line, Cal04 has a poor rate of infection (data not shown). Pigs vaccinated with whole inactivated IAV virus were detected by western blot. Validation of the anti-pig IgG secondary antibody as two separate repeats of the “positive control” serum (pig 5802) lit up NP strongly and M1 weakly. Several of the pigs of this study had a weak band indicating M1 weakly before and much more strongly post vaccination. M2 was detected post vaccination, but in the PR8 sample, not the Cal04 sample, further confirming that the pigs were free of M2 prior to vaccination with a band becoming visible after vaccination.

    3.4. Vaccine-challenge study humoral antibody responses

    Prior to vaccination, all pigs were confirmed IAV antibody negative, based on the commercial NP ELISA and in-house M2e-BSA ELISA. In addition, on dpv 28 and dpc 5, all pigs remained negative for IAV NP antibodies. In contrast, 6/10 M2e vaccinated pigs had seroconverted for M2e by dpv 14, 9/10 seroconverted by dpv 21, and all vaccinated pigs were M2e seropositive at the day of challenge. Data for the vaccine-challenge study are summarized in Fig. 4. None of the non-vaccinated pigs seroconverted to M2e (Fig. 4).

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    Fig. 4. M2e antibody response in the vaccine-challenge study. Seroconversion in pigs vaccinated with M2e-IM. Different superscripts (a,b) at a given day post vaccination indicate significant differences among groups. The cutoff for the ELISA is in an S/P ratio of 0.5 which is indicated by the green dotted line. Abbreviations: S/P, sample to positive ratio; dpv, day post vaccination; dpc, day post challenge. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    3.5. ELISpot

    Overall, the ELISpot results indicated minimal spots due to moderate hemorrhages interfering with the blotting process. Due to the small number of samples with appropriate results for each group, a statistical analysis was not done.

    3.6. Clinical signs

    Average daily weight gain was not different among the groups when measured from arrival to challenge (P = 0.91) or from challenge to necropsy (P = 0.98). IAV challenged pigs had increased respiratory scores compared to NEG-CONTROL pigs; however, this was not significantly different (data not shown). There was occasional mild cough in single pigs with no differences among groups. Rectal temperature data are summarized in Fig. 5. Overall M2e-IM-FLU pigs had the highest rectal temperatures which was not different from the POS-CONTROL pigs.

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    Fig. 5. Mean group rectal temperature after IAV challenge. Different superscripts (a,b) at a given dpc indicate significant differences among groups. Abbreviations: dpc, day post challenge.

    3.7. IAV nasal shedding after IAV challenge

    NEG-CONTROL pigs remained negative for IAV shedding throughout the study. In IAV-challenged pigs, IAV RNA shedding was evident from dpc 1 onwards. There were no significant differences among the two IAV challenged groups (Fig. 6).

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    Fig. 6. Nasal IAV shedding after IAV challenge. Different superscripts (a,b) at a given day post challenge (dpc) indicate significant differences among groups.

    3.8. Macroscopic lesions, microscopic lesions and IAV immunohistochemistry results on lung tissues at necropsy

    Macroscopic lung lesions were characterized by cranioventral, focally severe tan-red areas of consolidation typical of IAV infection. At necropsy there was a significant difference in overall gross lung lesion severity from challenged pigs to NEG-controls; however, there was no difference for vaccination status among IAV-infected pigs (Fig. 7A). Microscopic lesions were characterized by necrotizing bronchiolitis and tracheitis, suppurative bronchitis, exocytosis and interstitial pneumonia. Microscopic lesion severity and prevalence is summarized in Table 2.

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    Fig. 7. (A) Macroscopic lung lesions in percent. Score range is from 0 (no lesions) to 100% of the lung surface affected by lesions. (B) IAV staining in microscopic sections done by immunohistochemistry (IHC). Score range is from 0 (no staining) to 4 (diffuse moderate IAV staining). Different superscripts (a,b) at a given dpc indicate significant differences among groups.

    Table 2. Prevalence and severity of microscopic lung and trachea lesions among groups.

    Group Interstitial pneumonia* Peribronchiolar lymphoid cuffing** Bronchiolar necrosis** Suppurative bronchiolitis** Exocytosis** Trachea necrosis**
    NEG-CONTROL 0/10 (0)A 0/10 (0)A 0/10 (0)A 0/10 (0)A 0/10 (0)A 0/10 (0)A
    M2e-IM-FLU 9/10 (2.3 ± 0.3)B 9/10 (1.4 ± 0.3)B 9/10 (2.2 ± 0.3)B 9/10 (1.1 ± 0.2)B 3/10 (0.4 ± 0.2)A,B 4/10 (0.7 ± 0.3)A
    POS-CONTROL 10/10 (2.7 ± 0.1)B 10/10 (1.9 ± 0.2)B 10/10 (2.9 ± 0.1)B 10/10 (1.5 ± 0.2)B 6/10 (0.9 ± 0.3)B 8/10 (1.0 ± 0.4)A

    * Score range from 0 = normal to 6 = diffuse, severe.

    ** Score range from 0 = normal to 4 = severe.

    Different superscripts (A,B) among groups indicated a significant difference in group means (P < 0.5).

    IAV infected pigs also had moderate to high levels of IAV IHC staining with no differences between vaccinated and non-vaccinated pigs (Fig. 7B) whereas NEG-CONTROLs were IAV IHC negative.

    4. Discussion

    A vaccine is needed that improves cross-protection of pigs against multiple strains of IAV. In this study, an experimental vaccine using a highly conserved M2e peptide was evaluated in the pig model using the intramuscular administration route. Our hypothesis was that a single dose of M2e peptide vaccine would provide cross-protection against multiple IAV strains. Anti-M2e antibodies are thought to not neutralize the virus; however, they may prevent viral budding, enhance Fc-opsonization, complement activation, or mediate the killing of virally-infected cells using NK cells or macrophages through antibody dependent cell cytotoxicity (ADCC) [43]. Both P particle and large complex-based vaccines containing the M2e epitope have been produced in E. coli with high yield and stability [23], [24]. In mouse experiments, both vaccines were highly immunogenic and provided full protection against lethal IAV challenge [23], [24]. However, these vaccines were not successful in pigs, which may have been due to an insufficient dose. P-particle and complex based vaccines are difficult and expensive to produce at high concentrations. Therefore, these vectors were not further pursued. In addition, in the pilot study we also investigated the suitability of an intranasal vaccine but we could not see any measurable antibody responses in those pigs. Previously, it was found that aerosol administration of.

    a flu vaccine to pigs induced lung tissue-resident memory T cells and reduced lung pathology but not the viral load [44]. In another study, pigs were vaccinated with a novel vaccine delivery platform using mucoadhesive chitosan nanoparticles administered through the intranasal route [45]. Vaccinated pigs exhibited an enhanced IgG serum antibody and mucosal secretory IgA antibody responses in nasal swabs, bronchoalveolar lavage fluids, and lung lysates against H1N2, H1N1 and H3N2 IAV [45]. The intramuscular route is also consistent with current approved IAV vaccines in swine and thus, the goal here was to follow current vaccine platforms. Due to funding limitations, we could only go with one vaccination route in the main study and hence decided to go with the intramuscular route of vaccination.

    To assess the ability of the vaccine to induce anti-IAV antibodies, an M2e-based ELISA was developed and validated. While there are commercial M2e ELISAs, most are standardized for mice or humans. Initially, a commercially available avian M2e was purchased for this study. The homology between the avian M2e and the herein selected pig vaccine M2e was 98.2 % at the nucleic acid level. This was based upon 933 avian influenza virus (H5N1) sequences from GenBank used as the template for the avian M2e peptide. During the validation steps of the avian M2e ELISA, nonspecific cross-reactions were observed using serum samples from multiple studies (data not shown). When pig samples obtained at the start of the study and at dpv 28 were compared, cross-reactions were evident even in the samples obtained prior to vaccination. However, the NP-ELISA was used to confirm all pigs were negative. To further investigate this, an M2e-BSA was produced and tested to confirm the presence of anti-M2e antibodies in vaccinated pigs. In addition, a western blot assay using pdm09 M2e antigens ruled out the presence of detectable pre-existing M2e antibodies in the NEG-CONTROL pigs.

    To further assess the efficacy of M2e IM vaccination, a challenge study was conducted. Vaccinated pigs seroconverted to M2e/BSA whereas non-vaccinated pigs remained negative. Four weeks after vaccination, pigs were challenged with a pH1N1 US strain. Both vaccinated and non-vaccinated control pigs challenged developed fever and had moderate gross and microscopic lesions with no differences among groups suggesting that under the conditions of this study, the M2e vaccine did not protect pigs against challenge with pandemic H1N1 strain. This suggests that a humoral immune response, based only on the M2e protein, was not sufficient for protection. Measuring cellular immune responses against targets in pigs is notoriously difficult. In this study, sufficient, valid data was lacking to assess the cellular immune response in the pigs. Previous studies have reported enhanced disease and lung pathology upon challenge of pigs that had been vaccinated with conserved IAV antigens often in form of whole inactivated vaccines [46], [47]. This phenomenon has been defined as vaccine-associated enhanced respiratory disease (VAERD) [48]. In the present study, there was clearly no enhanced pathology due to presence of M2e antibodies. This is an important result because this means M2e peptide vaccines could have a potential to be safe if their efficacy can be improved.

    M2e adopts at least two converted conformations, and the intermolecular linker of M2e enhances conformational instability, limiting recognition by B cell receptors and production of high-level antibodies [49], [50]. Recently, a novel M2e vaccine expressed in a PCV2 vector has been reported [51]. Specifically, three M2e sequences derived from human, swine and avian IAV were inserted into the C-terminal of Cap protein to form nano vaccines [51]. Efficacy studies were done in mice indicating that the M2e closest to the surface of the nanoparticle induced the most efficient protection against IAV derived from corresponding species [49]. Previously M2e vaccines have been developed for dogs [52] and improved immune responses. Overall, the study outcomes indicate that a humoral immune response against M2e in pigs can be induced but is not protective.

    CRediT authorship contribution statement

    Tanja Opriessnig: Conceptualization, Formal analysis, Funding acquisition, Supervision, Writing – original draft, Writing -review & editing. Phillip C. Gauger: Funding acquisition, Supervision, Writing -review & editing. Patricia Filippsen Favaro: Formal analysis, Writing -review & editing. Gaurav Rawal: Formal analysis, Writing -review & editing. Drew R. Magstadt: Formal analysis, Writing -review & editing. Paul Digard: Formal analysis, Writing -review & editing. Hui-Min Lee: Formal analysis, Writing -review & editing. Patrick G. Halbur: Formal analysis, Supervision, Writing -review & editing.

    Declaration of competing interest

    The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


    We thank Professor Xavier Saelens for helpful advice and discussion over M2e peptide design, Dr Li Wang for support with the figure preparations and Dr Alessandra M.M.G. Castro for assistance with a part of the animal studies. We would also like to thank the animal care staff for excellent care and undergraduate, veterinary students and laboratory staff for their assistance with the sample collection, analysis and storage.


    This project was funded by the Iowa Livestock Health Advisory Council (ILHAC), United States. Brok Miller received a summer scholar stipend through the Iowa State University, United States, College of Veterinary Medicine Summer Scholars Research Program. PD was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) (award numbers BBS/E/D/20002173 and BBS/E/D/20002174) awarded to the Roslin Institute, University of Edinburgh, United Kingdom. For the purpose of open access, the author has applied a creative commons attribution CC-BY licence to any author accepted manuscript version arising from this submission.

    Data availability

    Data will be made available on request.


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