2-12 C mAb preparation
The anti-influenza HA1 human IgG1 mAb 2-12 C was produced by Absolute Antibody Ltd (Redcar, UK). It was dissolved in 25 mM histidine, 150 mM NaCl, and 0.02% Tween 20 (pH 6) diluent.
Influenza infection of pigs
All experiments were approved by the ethical review processes at the Pirbright Institute and Animal and Plant Health Agency (APHA) and were conducted in accordance with the UK government Animals (Scientific procedures) Act 1986, supported by project licence PP2064443. Seven influenza challenge experiments were carried out, three direct challenges and four contact challenges (Table 1). For all studies, 4–5-week-old female Large white x Landrace pigs were obtained from a commercial high-health status herd. The pigs were screened by ELISA for the absence of serum antibodies against A/swine/England/1353/2009 (pH1N1). In all experiments, animals acclimatized for at least 7 days and were randomized into different groups and pens using Excel by the animal services staff. Researchers processing the samples were only aware of the pig numbers, not the group assignments. The pathologists were blinded to the group allocation when assessing the samples for gross pathology during post-mortem examination and subsequently during the histopathological assessment. All conditions were kept the same between groups. Viral shedding was the main outcome variable for the studies. From previous experiments, the standard deviation in shedding is 0.8 log₁₀ pfu/ml. A reduction in mean viral shedding of 2 log10 pfu/ml is sufficient to show that treatment with the mAb has equivalent reduction to current influenza vaccines23,24. Using this difference and standard deviation a group size of five or six pigs is required to detect a difference between groups with >79% or 90% power respectively at 95% confidence (one-way ANOVA, six groups; Minitab 20). This design allows assessment of the effects of dose and delivery route with primary measures viral load as well as lung pathology.
First experiment (direct challenge I)
Animal experiment was conducted in APHA – ethical approval PP2064443-1-004, 6th August 2024. Twenty four pigs (average weight 10.8 kg) were randomized into four groups of six pigs as follows: 1) 2-12 C administered intravenously (IV) at 15 mg/kg into the ear vein, 2) 2-12 C (3 ml of 15 mg/ml, 1.5 ml per nostril) administered intranasally by mucosal atomization device (MAD, Medtree, UK), 3) 2-12 C (4 ml of 15 mg/ml) administered by aerosol using vibrating mesh nebuliser (VMN, Aerogen), and 4) untreated controls. For the 2-12 C mAb intravenous, MAD and VMN administration pigs were sedated with 1.5 mg/kg Zoletil (Virbac, UK) and 0.04 mg/kg medetomidine (Domitor, Orion Pharma, Finland). Twenty-four hours after 2-12 C administration, all animals were intranasally inoculated by MAD with 3 ×106 pfu of pH1N1 MDCK grown in 2 ml (1 ml per nostril) without sedation. Daily nasal swabs were collected for 4 days, and blood samples were collected at -1, 1-, 3-, and 4-days post infection (DPI). The pigs were humanely killed 4 days post pH1N1 infection with an overdose of pentobarbital sodium anaesthetic, confirmed by the permanent cessation of circulation. Lung pathology was assessed, and blood, bronchoalveolar lavage (BAL), and accessory lung were collected for assessment of viral load and antibody titres.
Second experiment (direct challenge II)
Animal experiment was conducted in APHA – ethical approval PP2064443-1-003v1 23rd Feb 2024. Fifteen pigs (average weight 11.6 kg) were randomized into three groups of five pigs as follows: 1) 2-12 C administered IV at 40 mg/kg following sedation as above, 2) 2-12 C at 25 mg/ml (4 ml) administered by VMN as above, and 3) untreated controls. Twenty-four hours after 2-12 C administration, all animals were intranasally inoculated by MAD with 3 ×106 PFU of pH1N1 in 2 ml (1 ml per nostril). Daily nasal swabs were collected for 4 days, and blood samples were collected at -1, 0, 1, 3, and 4 DPI. At 4 days post pH1N1 infection, the pigs were humanely euthanized with an overdose of pentobarbital sodium anaesthetic, confirmed by the permanent cessation of circulation. The lung pathology was assessed, and blood, BAL fluid, and accessory lung were collected.
Third experiment (contact challenge 6 days co-housing)
Contact challenges were performed in the high containment isolation unit at the Pirbright Institute at 19 °C and relative humidity of 55%—approved by animal facility team; named veterinary surgeon (NVS), named animal care and welfare officer (NACWO), animal facility manger, in house statistician, home office liaison contact (HOLC) and named information officer (HIO) officer, AR001352, 18th August 2023. Twenty pigs (average weight 12.2 kg) were split into two groups of ten donor pigs and ten recipient pigs. The recipient pigs were further randomized into two groups of 5 pigs as follows: 1) 2-12 C 14 mg/ml administered IV into the ear vein following sedation with a 1.5 mg/kg Zoletil and 3 mg/kg Stresnil (Elanco UK AH Limited), 2) untreated controls. The donor pigs were infected intranasally by MAD with 5 ×106 PFU pH1N1 (1 ml per nostril). After 24 h the five recipient 2-12 C pigs were put in contact with five donor pigs infected 24 h previously with pH1N1. Similarly, the five control untreated recipient pigs were put in contact in a separate room with five previously infected donor pigs. After 6 days of cohousing the donors were removed and culled. The recipient pigs were humanely culled 2 days later (8 days after cohousing) with an overdose of pentobarbital sodium anaesthetic, confirmed by the permanent cessation of circulation. Lung pathology was assessed, and samples of blood, BAL, and accessory lung tissue were collected for evaluation of viral load and antibody titers. Nasal swabs were taken daily from all pigs, and serum was collected at 2-, 5-, 7-, and 8- days post contact (DPC) from the recipient group only.
Fourth experiment (contact challenge 3 days co-housing)
Contact challenges were performed in the high containment isolation unit at the Pirbright Institute—approved by animal facility team (as above), AR001405, 29th April 2024. Twenty pigs (average weight 11.7 kg) were randomised and split into two groups of ten donor and ten recipient pigs. The recipient pigs were further randomized into two groups of five 1) 2-12 C administered IV at 25 mg/kg into the ear vein following sedation as in experiment 3 and 2) untreated controls. The donor group was infected with 5 ×106 PFU/ml pH1N1 virus. Twenty-four hours after 2-12 C administration or pH1N1 challenge, the recipients and donors were cohoused together. After 3 days of co-chousing the donor pigs were removed. The recipient pigs were humanely culled 6 days post contact with an overdose of pentobarbital sodium anaesthetic, confirmed by the permanent cessation of circulation. The lung pathology was assessed, and blood, BAL and accessory lung were collected. Nasal swabs were taken daily from all pigs, and serum was collected at 0-, 2-, 5-, and 6- DPC from the recipient group only.
Fifth experiment (contact challenge 3- and 2-days co-housing)
Contact challenges were performed in the high containment isolation unit at the Pirbright Institute—approved by animal facility team (as above), AR001423, 27th Sep 2024. Fifty pigs (average weight 10.4 kg) were split into two groups of twenty-five donor and twenty-five recipient pigs. The recipient pigs were randomized into four groups of five pigs as follows: 1) 2-12 C administered IV at 15 mg/kg into the ear vein, 2) 2-12 C administered by MAD (25 mg/ml, 1.5 ml per nostril) following sedation as in experiment 3, 3) 2-12 C administered by VMN (25 mg/ml, 3 ml), and 4) untreated controls. VMN and MAD administration as repeated after 48 h by the same route and dose. The 2-12 C administration was performed following sedation as in experiment 3. The donor group was infected with 5 ×106 PFU pH1N1. Twenty-four hours after 2-12 C treatment or pH1N1 challenge, each group of recipients was put in contact with five pH1N1 infected donors. Recipients and donors were cohoused together for 3 days, at which point the donor pigs were removed. The recipient pigs were kept for 3 more days and humanely killed 6 days post contact (labelled as Experiment 5a – 3 days co-housing). One untreated recipient group was cohoused with 5 donor pigs for 2 days only to compare the efficacy of contact infection between 2- and 3-days co-housing (labelled as Experiment 5b – 2 days co-housing). The recipient pigs were humanely culled 6 days post contact with an overdose of pentobarbital sodium anesthetic, confirmed by the permanent cessation of circulation. Lung pathology was assessed, and samples of blood, BAL, and accessory lung tissue were collected for evaluation of viral load and antibody titers. Nasal swabs were taken daily from all pigs, and serum was collected at 0-, 2-, 5-, and 6-DPC from the recipient group only.
Sixth experiment (contact challenge 2- days co-housing)
Contact challenges were performed in the high containment isolation unit at the Pirbright Institute – approved by animal facility team (as above), AR001465, 8th January 2025. Forty pigs (average weight 16 kg) were split into two groups of 20 donors and 20 recipients. The donor group were randomised into 4 groups of five animals. One group was given 2-12 C intravenously at 10 mg/kg. Twenty-four hours later all four groups of donors were intranasally inoculated using MAD with pH1N1 with the following doses: 1) 1 ×106 pfu; 2) 1 ×104 pfu; 3) 1 ×103 pfu. 4) The group treated with 2-12 C was inoculated with 1 ×106 pfu pH1N1. Twenty-four hours after the pH1N1 challenge, the recipients and donors were cohoused together. After 2 days of co-chousing the donor pigs were removed. The recipient pigs were humanely culled 6 days post contact with an overdose of pentobarbital sodium anaesthetic, confirmed by the permanent cessation of circulation. Lung pathology was assessed, and samples of blood, BAL, and accessory lung tissue were collected for evaluation of viral load and antibody titers. Nasal swabs were taken daily from all pigs, and serum was collected at 0-, 2- DPC from the 2-12 C-treated group only.
Seventh experiment (direct challenge, different volumes of viral inoculum)
The direct challenges were performed in the high containment isolation unit at the Pirbright Institute – approved by animal facility team (as above), AR001496, 18th March 2025. This was an exploratory pilot study with 3 or 4 pigs per group. Ten pigs (average weight 10.6 kg) were randomly allocated into three groups and infected with 104 pfu pH1N1 delivered either in 1 ml (3 pigs), 0.5 ml (3 pigs) and 0.25 ml (4 pigs). Daily nasal swabs were collected for 4 days. At 4 days post pH1N1 infection, the pigs were humanely euthanized with an overdose of pentobarbital sodium anaesthetic, confirmed by the permanent cessation of circulation. Lung pathology was assessed, and samples of blood, BAL, and accessory lung tissue were collected for evaluation of viral load and antibody titers.
Clinical signs, including temperature, coughing, breathing status, appetite, nasal discharge, and behavioural change, were monitored daily after the pH1N1 challenge in all experiments. The observed signs were mild, and none of the pigs developed moderate or severe disease.
Pathological and histopathological examination of lungs
Gross pathology and histopathological analysis were performed at postmortem as described previously25. In brief, the lungs were removed, and the dorsal and ventral aspects of the lungs were photographed. Macroscopic pathology was blindly scored by a veterinary pathologist as previously reported26. Lung tissue samples were taken from the right cranial, middle, and caudal lung lobes from the right side of the lung and immersed into 10% neutral-buffered formalin for histological processing. The formalin-fixed tissue was embedded in paraffin wax, cut into 4-mm sections and routinely stained with H&E and immunohistochemistry against influenza A virus nucleoprotein (NP). Lung histopathology and IHC staining were assessed by a veterinary pathologist blinded to the treatment group. Pulmonary histopathology was scored (“Morgan”) using five parameters (airway inflammation, alveolar exudates, septal inflammation, necrosis of the bronchiolar epithelium and perivascular/bronchiolar cuffing) using a five-point scale of 0–4, the sum of which gave a total score ranging from 0–20 per lobe27. A mean score for the three lung lobes was calculated for each animal. Another scoring system “Iowa” was used to score the individual lung lobes and also considering the amount of NP present in the samples28.
Virus titration
Viral titres in nasal swabs, accessory lung lobe and BAL fluid were determined by plaque assay using Madi-Darby canine kidney (MDCK) cells. Samples were serially diluted in Dulbecco’s Modified Eagle Medium (DMEM), before being overlayed on confluent MDCK cells. After incubation at 37 °C at 5% CO2 for 1 hour, plates were washed with PBS and overlayed with medium consisting of 1× Minimum Essential Medium Eagle (Sigma-Aldrich). 0.3% BSA, 2.86 mM L-Glutamine, 0.3% sodium bicarbonate, 14 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.007% dextran, 100 IU/ml penicillin, 100 μg/ml streptomycin and 0.6% agar (Oxoid). After a further incubation at 37 °C at 5% CO2 for 72 h, the overlay was removed and 0.1% crystal violet (Sigma-Aldrich) in 20% methanol (Sigma-Aldrich) was used to stain the cells and count the plaques. Plaque forming units per mL (PFU/ml) were calculated as the average number of plaques divided by the dilution factor.
Tissue sample processing
Serum was collected using BD Vacutainer SST™ Serum Separation Tubes which were centrifuged for 15 mins at 1000 × g. Two nasal swabs, one from of each nostril, were taken at the time points indicated and placed in 2 ml of virus transport medium (VTM) consisting of medium 199 (Sigma-Aldrich) supplemented with 0.5% BSA, 100 IU/ml penicillin, 100 μg/ml streptomycin, 0.035% sodium bicarbonate, 25 mM HEPES and 0.25% mg/ml nystatin. The samples were vortexed, centrifuged to remove debris, and stored at −80 °C for subsequent virus titration.
BAL fluid was collected using 0.3% BSA in PBS which was added into the left lung and then collected in falcon tubes. BAL fluid was then centrifuged at 500 × g for 5 mins, with supernatant removed and frozen until use. Accessory lung lobes were collected from all pigs at postmortem and frozen at −80 °C. After thawing, 2 g of tissue, extracted uniformly from across the entire lobe, was added to 9 ml of RPMI media and homogenized using a Miltenyi Cell Dissociator. An additional 9 ml of RPMI media was then added before the cell suspension was centrifuged at 500 × g for 5 min. The supernatant was removed, aliquoted and frozen for analysis of viral load by plaque assay and antibody titres.
ELISA and microneutralisation
Serum, BAL fluid and nasal swab antibody titres were assessed by ELISA against recombinant HA protein of A/Eng/195/2009 hemagglutinin as before29. Briefly, serum, BAL fluid and nasal swabs were heat inactivated at 56 °C for 30 mins and diluted in buffer (0.05% Tween 20 in PBS). HA coated plates were blocked using 4% (w/v) milk powder (Marvel) in PBS containing 0.05% Tween 20 (Sigma-Aldrich) before the sera samples were incubated with HA-coated plates in duplicate for 1 hour at RT for serum and BAL fluid and overnight for nasal swabs. The plates were washed twice and incubated with HRP conjugated goat anti-human IgG-Fc antibody HRP (Bethyl laboratories, A80-304P) for 1 hour at RT and developed using 3,3′,5,5′-tetra-methylbenzidine (TMB) substrate. The plates were read at 450 nm and 630 nm (reference wavelength) with the Absorbance Microplate Reader (BioTek, Swindon, UK). The serum concentration was interpolated from the standard curve using a sigmoidal four-parameter logistic curve fit for the log of the concentration.
Neutralising antibody titres were determined in serum and BAL fluid using microneutralisation assay as before16. In brief, serum and BAL fluid that were heat inactivated at 56°C for 30 mins and were serially diluted before being incubated with an equal volume of pH1N1. After 2 h, MDCK SIAT-1 cells at 3 ×104 cells per well were added and then incubated for a further 18 h. The cell monolayer was fixed with 4% paraformaldehyde and permeabilised with 0.05% Triton-X100 and 20 mM glycine and then stained with mouse anti-NP IAV IgG1 (Clone AA5H, Bio-Rad Laboratories) followed by goat anti-mouse HRP secondary antibody (DAKO). After addition of the TMB substrate, the reaction was stopped with 1 M sulfuric acid, and absorbance was measured at 450 and 570 nm (reference wavelength) on the Absorbance Microplate Reader (BioTek). The 50% inhibition titre was defined as the final dilution of serum that caused ≥50% reduction in NP expression.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 10.0.2 (GraphPad software, San Diego, CA, USA). Data sets were first assessed for normality and then subjected to either a t-test or one-way or two-way ANOVA test and a post-hoc Tukey’s multiple comparisons test or Dunnett’s multiple comparisons test, or a Kruskal-Wallis test and a post-hoc Dunn’s multiple comparisons test if they did not pass the normality test. Significant differences were presented on each graph (*p
