A live attenuated NS1-deficient vaccine candidate for cattle-origin influenza A (H5N1) clade 2.3.4.4.b viruses

Biosafety

All in vitro and in vivo studies involving highly pathogenic and low pathogenic avian influenza virus (HPAIV and LPAIV, respectively) H5N1 were conducted at biosafety level (BSL) 3 and animal BSL3 (ABSL3) laboratories at Texas Biomedical Research Institute (Texas Biomed). These experiments were approved by both the Texas Biomed Institutional Biosafety (IBC) and the Animal Care and Use (IACUC) committees.

Cells

Madin-Darby canine kidney (MDCK), human embryonic kidney (293 T), African green monkey kidney (Vero), and MDCK cells expressing GFP and firefly luciferase (FFluc) under the control of the interferon (IFN) β promoter (MDCK pIFNβ-GFP/IFNβ-FFluc)27 were cultured in Dulbecco’s modified Eagle medium (DMEM) (Invitrogen, USA) complemented with 10% fetal bovine serum (FBS) and 1% PSG (penicillin, 100 U/mL; streptomycin, 100 µg/mL; L-glutamine, 2 mM). Cultured cells were kept at 37 °C in a humidified 5% CO2 incubator.

Plasmids and viruses

The recombinant low pathogenic human influenza virus A/Texas/37/2024 H5N1 (LPhTX) was generated as previously described28 and propagated in MDCK cells using infection media (DMEM containing 1% P/S/G and 0.2% BSA) in the presence of 1 μg/mL of L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Sigma–Aldrich, USA).

The pHW-hTX_NS plasmid28, which expresses the NS1 and nuclear export protein (NEP) of A/Texas/37/2024 H5N1, was used as a template to remove the NS1-coding sequence. Briefly, 2 μl (5 ng/μl) of pHW-hTX_NS were combined with 5 μl of 10X Pfu reaction buffer, 1 μl of 10 mM dNTP mixture (0.2 mM of each), 1.5 μl of 50 mM MgCl2 (1.5 mM), 2 μl of forward primer (F: GTTAAGCTTTCAGGACATACTGATGAGGATGTCAAAAATGCAATTGGGGTCCTCAT) (40 pmoles), 2 μl of reverse primer (R: CTGAAAGCTTAACACAGTGTTGGAATCCATTATGTTTTTGTCACCCT) (40 pmoles), and 1 μl of PfuTurbo DNA Polymerase (2.5 U/μl) (Invitrogen, USA). The total volume was adjusted to 50 μl using nuclease-free water (Ambion, USA). The reaction was preceded by a pre-denaturation at 95 °C for 2 min, followed by 35 PCR cycles (95 °C for 30 s for denaturation, 56 °C for 30 s for annealing, and 72 °C for 3 min for extension), and a final extension step at 72 °C for 10 min. The PCR product was purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, USA). The resulting PCR product was then digested with HindIII (NEB, USA) to create the pHW-hTX-dNS1 plasmid.

To generate LPhTXdNS1, the seven ambisense pHW2000 plasmids containing the PB2, PB1, PA, monobasic HA, NP, NA, and M segments of LPhTX, and the pHW-hTX-dNS1 plasmid (1 μg each) were co-transfected into a 293 T/MDCK cell co-cultures (3:1 ratio) using Lipofectamine™ 3000 Transfection Reagent (ThermoFisher Scientific, USA), according to the manufacturer’s protocol. After 24 h, the transfection medium was replaced with 1 mL of Opti-MEM media containing 1% PSG and 0.2% bovine serum albumin (BSA) and the culture was incubated in a 5% CO2 incubator. Twelve hours later, an additional 1 mL of Opti-MEM media containing 1% PSG, 0.2% BSA, and 2 μg/mL of TPCK-treated trypsin was added to each well. After 48-72 h, cell culture supernatants were harvested and centrifuged at 2500 rpm for 5 min at 4 °C. A portion of the collected supernatant was used to infect MDCK monolayers in T-75 cell culture flasks in infection media (DMEM with 1% PSG, 0.2% BSA, and 2 μg/mL of TPCK-treated trypsin). The rescued LPhTXdNS1 was then aliquoted and stored at −80 °C.

Viral stock was confirmed by whole genome sequencing using the MinION platform (Oxford Nanopore Technologies) in comparison to the generated LPhTX strain. Briefly, viral RNA was extracted using the QiAamp Viral RNA Mini Kit (Qiagen, USA), and sample libraries were prepared with the Native Barcoding Kit 24 V14 (SQK-NBD114.24, Oxford Nanopore Technologies). Sequencing was performed on R10.4.1 Flow Cells (FLO-MIN114, Oxford Nanopore Technologies) according to the manufacturer’s instructions. Following nanopore sequencing, the initial read quality statistics were evaluated using n50 v1.7.0, followed by quality filtering with nanoq v0.10.029. Reads were trimmed once average PHRED quality scores dropped below Q < 10. In addition, we removed the first and last 15 base pairs of each read. After trimming, reads shorter than 500 base pairs were completely removed. Quality-trimmed reads were polished using the self-correcting algorithm LoRMA v0.4.230 by comparing batches of 100 overlapping reads. Polished reads were aligned to the H5N1 LPhTX reference genome using minimap2 v2.28-r120931 with the -x map-ont parameter to optimize mapping of error-prone, long Nanopore reads. Alignment statistics were calculated using SAMtools flagstat v1.2132, and genome-wide coverage was estimated using MosDepth v0.3.1033. We used LoFreq v2.1.534 to evaluate indel quality and identify sequence variants. For variant calling, we capped the maximum allowable read depth at 10,000x (--max-depth) and disabled LoFreq’s default filters. Instead, we applied custom filtering criteria, retaining only variants supported by at least 25% of reads and located in regions with a minimum read depth of 100x.We also removed indels caused by homopolymer runs of three or more bases. For reproducibility, all analyses were completed on worker nodes containing 192 cores and 1 Tb of memory at the Texas Biomedical Research Institutes high-performance computing center. All code necessary to complete the genetic analysis is available at GitHub (https://github.com/nealplatt/h5n1_highpathcow_2025-03-17). Sequence data is archived under BioProject accession code “PRJNA1242379”.

A recombinant A/Texas/37/2024 H5N1 containing a non-structural (NS) segment where the C-terminus of NS1 was fused to Nanoluciferase (NLuc) was engineered as previously described35. The recombinant NS segment was synthesized de novo (Bio Basic, USA) with the appropriate restriction sites for subcloning into the ambisense plasmid pHW200 to generate the pHW-H5N1-NS_AgeI/NheI plasmid. The recombinant NS segment contained the NS1 open reading frame (ORF) without stop codons or splice acceptor sites, followed by AgeI and NheI restriction sites, the porcine teschovirus-1 (PTV-1) 2 A autoproteolytic cleavage site (ATNFSLLKQAGDVEENPGP) and the entire ORF of the nuclear export protein, NEP36. The Nluc ORF was cloned using the AgeI and NheI sites, into pHW-H5N1-NS_AgeI/NheI to generate the pHW-H5N1-NS_Nluc for virus rescue. Plasmid constructs were confirmed by DNA sequencing (Plasmidsaurus, USA). Recombinant A/Texas/37/2024(H5N1) virus expressing NS1_Nluc (HPhTX-Nluc) was rescued as previously described28,37,38,39,40. HPhTX-Nluc was then plaque purified and amplified on MDCK cells to generate the viral stock.

Plaque assay and immunostaining

MDCK and Vero cells were seeded at a density of 106 cells per well in 6-well plates and incubated overnight at 37 °C in a humidified 5% CO2 incubator. The following day, the confluent MDCK monolayers were infected with 10-fold serial dilutions of the virus for 1 h at 37 °C. After viral adsorption, the cell monolayers were overlaid with post-infection media containing agar and incubated at 37 °C in a humidified 5% CO2 incubator. At 24 (immunostaining) and 72 (plaque assay) hours post-infection (hpi), cells were fixed for 24 h with 10% neutral buffered formalin. For staining and visualization of viral plaques with crystal violet, 1 mL of 0.1% crystal violet solution was applied to each well for 5 min at room temperature (RT), followed by rinsing with tap water. For immunostaining, cells were permeabilized with 0.5% Triton X-100 in PBS for 15 min at RT and stained with the mouse monoclonal antibody (MAb) HT103 against influenza viral nucleoprotein (NP)41 and the Vectastain ABC kit (Vector Laboratories, USA), following the manufacturer’s protocol. After immunostaining, the plates were scanned and photographed using the ChemiDoc MP Imaging System.

Western blotting

Vero cell monolayers (6-well plate format, 106 cells/well, triplicates) were infected with LPhTX and LPhTXdNS1 at multiplicity of infection (MOI) of 2. At 12 hpi, cells were washed with ice-cold PBS and treated with ice-cold NP40 lysis buffer (Thermo; completed with protease inhibitors cocktail). Cells were lysed on ice for 30 min, scrapped off and transferred to 1.5 mL Eppendorf tubes. Cells were centrifuged at 15,000 rpm for 15 min at 4 °C. The cell lysate supernatants were then mixed with 2x working lithium dodecyl sulfate (LDS) sample buffer, supplemented with 20% β-mercaptoethanol and incubated at 98 °C for 5 min before loading into SDS-PAGE gels. Following sample running, gels were transferred into nitrocellulose membranes. Subsequently, membranes were incubated in 5% non-fat dry milk powder in PBS with 0.05% Tween 20 blocking buffer (PBST) (Sigma, USA). Blocked membranes were probed overnight at 4 °C with primary antibodies against PB1 (Clone F5-46, BEI Resources), PA (Clone 1F6, BEI Resources), NP (HT103)41, NS1 (pAb, 1-73SW)15, and β-actin (clone AC-15; A1978; Sigma, USA) as a house keeping protein. After overnight incubation, membranes were washed 3x for 5 min each with 5 mL of PBST. After washing, membranes were incubated with HRP-conjugated secondary antibodies for 2 h at RT. After 3x washes for 5 min with 5 mL of PBST, chemiluminescence-generated protein bands were visualized using a SuperSignal ECL substrate kit (ThermoFisher, USA) following manufacturer’s recommendations.

Viral replication kinetics

Vero cell monolayers cultured in 6-well plates (106 cells per well, triplicates) were infected with the specified viruses at a MOI of 0.001 and plates were kept at 37 °C or 33 °C in a humidified 5% CO2 incubator for 1 h to allow viral adsorption. After viral adsorption, the inoculum was removed and infected cell monolayers were washed 3x with PBS to eliminate any unadsorbed viral particles. Cell monolayers were then supplemented with 3 mL of infection medium containing 2 ug/mL of TPCK-treated trypsin and incubated at 37 °C in a humidified 5% CO2 incubator. Aliquots of 200 µL were taken from the cell culture supernatants at 12, 24, 48, and 72 hpi and replaced with an equal volume of fresh infection medium. Viral titers in the collected samples were determined using plaque assays and immunostaining, as previously described28,42.

Mice experiments

Female C57BL/6 J mice (n = 13/group) obtained from The Jackson Laboratory (JAX, USA) were anesthetized intraperitoneally (i.p.) by a cocktail of Ketamine (100 mg/mL) and Xylazine (20 mg/mL). Anesthetized mice were infected intranasally (i.n.) with the indicated viral doses in a total volume of 50 μl PBS. On 2-, 4-, and 6-days post-infection (DPI), 4 mice in each group were subjected to In Vivo Fluorescence Imaging (IVIS), if required, and were subsequently euthanized intraperitoneally using a lethal dose of Fatal-Plus ( >100 mg/kg) to collect lung, nasal turbinate, and brain tissues. Half of the lung and brain organs were fixed in 10% neutral buffered formalin solution for histopathology and immunohistochemistry (IHC) analyses, and the other halves were homogenized in 1 mL of PBS using a Precellys tissue homogenizer (Bertin Instruments, USA) for viral titration. Tissue homogenates were centrifuged at 10,000 xg for 5 min and the supernatants were used to determine viral load by plaque assay and investigate the presence of secreted cytokines/chemokines. The remaining 5 mice/group were monitored for 14 days for body weight changes (morbidity), and survival rates (mortality). Mice that experienced a weight loss exceeding 25% of their original weight were humanely euthanized with carbon dioxide in accordance with the approved institutional protocol by the Animal Care and Use (IACUC) committee.

Hemagglutination inhibition (HAI) assay

Sera were collected weekly from both mock-vaccinated and vaccinated mice (n = 5/group). The sera were treated with receptor-destroying enzyme (II) (RDE-II, Denka Seiken, USA) for 20 h at 37 °C. The RDE-II enzyme was inactivated by heating at 56 °C for 30 min. After treatment, 12.5 µl of the sera was added in triplicate to 96-well plates and mixed with 12.5 µl of diluted LPhTX, containing 4 hemagglutinating (HA) units. The plates were incubated for 30 min at RT. Then, 25 µl of 1% turkey red blood cells (RBCs) was added to each well, and after 45 min HAI was determined as the highest dilution of antiserum that completely inhibited hemagglutination43.

Multiplex cytokine assay

Cytokines and chemokines (IFN-α, IFN-β, IFN-γ, IL-6, CCL2, and CCL5) were measured using a multiplex mouse ProcartaPlex assay (ThermoFisher Scientific), following the manufacturer’s instructions. Briefly, lung homogenates were first centrifuged at 10,000 x g for 5 min to remove debris and diluted 1:2 in Universal Assay Buffer. The assay was conducted in the ABSL3 laboratory, and samples were decontaminated by incubating overnight in 1% formaldehyde solution before being analyzed using a Luminex 100/200 System using xPONENT v4.3.309.1 software. The analysis parameters were as follows: gate range from 7500 to 25,000, 50 μl sample volume, 50 events per bead, a sample timeout of 60 s, and standard PMT settings.

Histopathology and immunohistochemistry (IHC)

Mice lung and brain tissues were collected at necropsy, fixed in 10% neutral buffered formalin and processed in a Tissue Tek VIP tissue processor, where they were dehydrated through a series of graded alcohols, cleared with a 50:50 absolute alcohol/xylene mixture and two changes of xylene, and then infiltrated with paraffin wax using ParaPro™ XLT infiltration and embedding media (StatLab, USA). The paraffin blocks were sectioned at 4 microns thick using a Microm HM325 rotary microtome and mounted on microscope slides using a flotation water bath set between 46 °C and 48 °C. The slides were then placed in a Varistain Gemini automated slide stainer for H&E staining, followed by drying in heating stations before the staining program began. The deparaffinization of tissue sections was done with three changes of xylene, two changes of absolute alcohol, and two changes of 95% alcohol, followed by rinsing in distilled water. Hematoxylin staining was conducted with Reserve Bluing Reagent (StatLab, USA), with excess stain removed using High-Def solution (StatLab, USA). The tissue sections were then stained with Eosin (StatLab, USA), dehydrated in three changes of alcohol, a 50:50 absolute alcohol/xylene mixture, and cleared in three changes of xylene before mounting them in coverslips. After staining, the tissue sections were examined by a board-certified veterinary pathologist in a blinded fashion. For IHC, tissue sections were cut at 4 microns, mounted onto positively charged slides, and allowed to air dry overnight. The slides were then processed using the Discovery Ultra IHC/ISH automatic stainer to detect avian influenza virus. Deparaffinization was performed using Discovery Wash (Roche, USA). Cell conditioning was done with Discovery CC1 (Roche, USA) at 95 °C for 64 min. Endogenous peroxidase was blocked with Discovery Inhibitor (Roche, USA) for 8 min. Slides were then incubated with a rabbit polyclonal antibody against IAV NP (Invitrogen, USA) at a 1:1500 dilution for 1 h at RT, followed by detection with an anti-rabbit HQ (Roche, USA) for 8 min, and anti-HQ HRP (Roche, USA) for 8 min at 36 °C. Influenza NP was visualized using ChromoMAP DAB (Roche, USA). The slides were counterstained with Hematoxylin (Roche, USA) and blued using Bluing Reagent (Roche, USA). All HE stained and IHC stained slides were scanned using Zeiss axio scan. Z1 whole slide scanner at ×20 magnification. The digital slides were analyzed using an AI optimized tissue classifier in HALO v4.0 software (Indica Labs, USA) for absolute quantification of the percentage of lung affected and percentage of lung staining for viral antigen.

Statistical analyses

All graphs, calculations, and statistical analyses were performed using GraphPad Prism software version 9.5.1 (GraphPad Software, LLC, USA). No assumptions of equal variance or sphericity were made for group comparisons. Normality of residuals was checked for all group comparisons and log10 transformations were applied when the assumption of normality was not met. Growth kinetics were analyzed using a two-way repeated measure ANOVA with Geisser-Greenhouse correction. Post-hoc comparisons were performed using the Šídák method. Differences in FFluc expression were evaluated using Welch’s one-way ANOVA, followed by multiple comparisons using Dunnett T3 method. Viral replication and titer data were analyzed using mixed-effects ANOVA, whereas cytokine expression data were analyzed using a two-way ANOVA. Dunnett’s multiple comparisons test was used to compare groups within each time-point. Post-hoc testing was conducted with Dunnett’s multiple comparisons test. Differences in survival curves were analyzed using a Log-rank test.

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