MHC class II functions as a host-specific entry receptor for representative human and swine H3N2 influenza A viruses

Domain: neuroscience
Published: 2026-02-10
Editorial outcome: major_revision

Abstract
Influenza A viruses (FLUAV) utilizes sialic acid to enter host cells via the envelope’s hemagglutinin (HA), and its affinity for host-specific sialic acid linkage configuration is a major host range determinant. However, some FLUAV subtypes (H17, H18, H19) use the major histocompatibility complex class II (MHC II) as an entry receptor instead of sialic acid (SA), challenging our knowledge about FLUAV tropism and interspecies transmission potential. Here, we show that H3N2 viruses can use MHC II as an alternative entry receptor in a host-specific manner, and adaptation of human viruses to pigs increases affinity for the MHC II swine leukocyte antigen (SLA). By using two prototypic human-seasonal (hVIC/11) and swine-adapted (sOH/04) H3N2 viruses we found that expression of the human (HLA) but not the swine MHC II conferred replication of hVIC/11 in desialylated, non-susceptible cells. Further, expression of SLA in non-susceptible cells conferred susceptibility to infection by sOH/04. Introduction of point mutations near the hVIC/11 HA receptor-binding site (RBS) allowed the use of both human and swine MHC II. Our findings revealed that MHC II can serve as a sialic acid-independent entry receptor to H3N2 FLUAV in a host-specific manner, with potential implications for the viral pathogenesis and adaptation to a new species.

Introduction
Sialic acid (SA) is the canonical receptor used by influenza A viruses (FLUAV). These SAs on the cell surface are recognized by the viral hemagglutinin (HA) protein to promote entry and begin the replication cycle1,2. The SA structure is linked to FLUAV host range since avian-origin FLUAVs mainly recognize SA linked to galactose by an α2,3 linkage (α2,3-SA) while mammalian-origin FLUAVs recognize α2,6-linked SA (α2,6-SA)3. Therefore, mutations in the HA protein in or near the receptor-binding site (RBS) can modify the binding preference of the virus4–6 and may lead to a switch in the host specificity3. By contrast, recently discovered bat-origin H17 and H18 HA subtypes, as well as duck-origin H19, were shown to exclusively use the Major Histocompatibility Complex class II (MHC II) as an entry receptor7–9. This interaction is completely independent of SA as multiple glycan array studies have shown H17-H19 fail to bind to all glycans tested9,10. Interestingly, H17 viruses displayed a species-specific affinity for MHC II in which the human leukocyte antigen isotype DR (HLA-DR) allowed efficient infection of non-permissive cells compared to cells expressing the swine homologue (SLA-DR)8. Conserved residues in the α1, α2, and β1 domains of the HLA-DR molecule were shown to be critical for H18N11 infection of human cells11. More recently, human H2N2 and related avian H2N2 FLUAV were shown to possess dual receptor specificity for SA and MHC II, with entry via MHC class II being independent of sialic acid12. This suggests that MHC II could play a role in the host range of FLUAV, although the ability of other SA-binding HA subtypes to use MHC II as an entry receptor remains unknown.

Pigs are a natural host of FLUAV and have historically been pointed out as important intermediary hosts for the generation of viruses with pandemic potential13. Frequent spillover of FLUAV between humans and swine, followed by subsequent evolution of a few of these strains in the new host, has impacted the epidemiology of circulating strains in both species14,15. We previously showed that adaptation of a human-origin H3N2 virus to pigs selected for a single HA mutation (A138S, H3 numbering) that increased affinity for porcine alveolar macrophages (PAMs) in vivo compared to the original HA16. This ultimately led to apoptosis and depletion of this cell population in the lungs of infected pigs. Additionally, we have observed that a swine (sOH/04) but not a human-adapted (hVIC/11) H3N2 virus can efficiently infect alveolar macrophages in vivo17,18. Considering that PAMs express high quantities of MHC II on the cell surface, we investigated the ability of hVIC/11 and sOH/04 to use HLA-DR or SLA-DR as receptors to initiate infection. We found that both viruses can use MHC II as an entry receptor in addition to SA in a host-specific manner. Moreover, the introduction of point mutations in the hVIC/11 HA RBS changed the virus host range and allowed utilization of the SLA-DR. These mutations also affected SA binding, suggesting that the MHC II-HA interaction is mediated by the RBS or its surroundings and that mutations in or near this pocket have a dual impact on the specificity of the virus for SA and MHC II.

Results
MHC II is needed for the infection of porcine alveolar macrophages (PAMs) by swine-adapted H3N2 FLUAV in the absence of SAs
To investigate whether SLA-DR-mediated entry plays a role in FLUAV infection in vivo, we leveraged samples from a previous study16 that showed significant tropism and replication differences between human-origin (hVIC/11) and swine-adapted (sOH/04) FLUAVs in the lungs of pigs. Analysis of lung samples from sOH/04-infected pigs revealed a strong colocalization between SLA-DR and FLUAV HA antigens, suggesting that SLA-DR+ cells might be susceptible to infection with the swine-adapted FLUAV in vivo (Fig. 1a). hVIC/11 was not included in the analysis as it fails to infect swine lungs16. However, it cannot be ruled out that the co-localization observed was due to PAMs’ intrinsic phagocytic activity. To further investigate the role of MHC II during FLUAV infection, we performed infections using alveolar macrophages collected and stored from naïve pigs in a previous study16. Since SA is the primary receptor used by H3N2 FLUAV, depletion of these receptors from PAMs was needed to evaluate the role of MHC II during FLUAV entry. Two methods were evaluated to remove SA on the cell surface: chemical oxidation and enzymatic SA removal with the NanI neuraminidase from C. perfringens, known to cleave both 2,3- and 2,6-Neu5Ac19 and Neu5Gc20. SA oxidation with sodium periodate (NaIO4) was highly cytotoxic (Supplementary Fig. 1a) while SA removal using neuraminidase treatment showed little cytotoxicity (Supplementary Fig. 1b). After a 24-h incubation with 100 mU/mL of neuraminidase (100% cell viability), most of α2,3 and α2,6 SA was successfully removed from PAMs (Fig. 1b and Supplementary Fig. 1c). Further, the neuraminidase treatment did not affect SLA-DR expression in these cells (Fig. 1c). Inoculation of untreated PAMs with sOH/04 and hVIC/11 resulted in replication of both viruses, although hVIC/11 displayed lower titers overall compared to sOH/04 (Fig. 1d, e), particularly at 48 h post infection (hpi). Neuraminidase treatment partially reduced replication of sOH/04 and hVIC/11. Infection of PAMs was significantly reduced only when both SA and SLA-DR were made unavailable by a neuraminidase treatment coupled with an anti-SLA-DR antibody incubation, in a dose-dependent manner (Fig. 1f). Incubation with a control antibody (anti-GFP) did not affect replication in desialylated PAMs (Fig. 1g). These results suggest that FLUAV can infect PAMs in a SA-independent manner, and these infections might be dependent on MHC II availability.

Transient expression of MHC II in desialylated cells enables FLUAV infection and replication
To confirm that FLUAV uses MHC II as an entry receptor, we transfected HEK-293T cells with plasmids encoding the alpha and beta chains of either HLA-DR or SLA-DR. HEK-293T cells were selected due to their high transfection efficiency21. Transient expression of MHC II was demonstrated by immunofluorescence analysis (Supplementary Fig. 2a). Subsequently, transfected cells were treated with exogenous neuraminidase (Supplementary Fig. 2b), and after a 24-h neuraminidase treatment, both α2,6-SA and α2,3-SA were detected on the cell surface (Supplementary Fig. 3). MHC II+ HEK-293T cells bearing or lacking SAs were inoculated with sOH/04 and hVIC/11. Under SA-depleted conditions, FLUAV only infected MHC II-expressing cells (Fig. 2a). This was further confirmed by flow cytometry (Supplementary Fig. 4a). Moreover, neuraminidase treatment completely prevented FLUAV infection when cells were transfected with an empty plasmid (Supplementary Fig. 4a, b). Levels of FLUAV-positive cells among those bearing SA were similar regardless of MHC II expression (Supplementary Fig. 4c). When only MHC II-expressing cells were considered (Fig. 2b, c), we found a clear trend in which hVIC/11 preferably infected HLA-DR-expressing cells. Similarly, transient expression of SLA-DR in desialylated cells significantly increased the affinity of sOH/04 to higher levels than when HLA-DR was present. Quantitatively, hVIC/11 infected 17.9% of HLA-DR+ cells vs 5.1% of SLA-DR-expressing cells (Fig. 2d). On this line, sOH/04 preferentially infected the SLA-DR+ cells in which 22.7% of cells were infected, contrasting with the 6.7% of infected cells when HLA-DR was present (Fig. 2d). Control infections performed in untreated cells or desialylated cells transfected with an empty plasmid supported that the transfection process alone did not allow FLUAV infection of non-sialilated cells (Fig. 2e). Notably, sOH/04 infected a higher percentage of cells when SLA-DR was the only available receptor compared to conditions where only SA was present (Fig. 2e, p=0.02). Alignment of the HLA-DR and SLA-DR alpha and beta chains showed multiple amino acid differences between the two homologue genes that could account for this effect (Supplementary Fig. 5). As a negative control, we used the 2009 pandemic H1N1 A/California/04/2009 virus (pH1N1), a subtype previously shown not to use MHC II as an entry receptor12. pH1N1 successfully infected untreated HEK-293T cells but failed to infect desialylated HEK-293T (Supplementary Fig. 6a), even when HLA-DR or SLA-DR were expressed (Supplementary Fig. 6b, c). After recognition of SA, FLUAV is internalized in endosomes that are further acidified, allowing the virus particle disassembly and genome release into the cytoplasm22. Therefore, we evaluated if FLUAV follows a similar route after interacting with MHC II. Prevention of endosomal acidification by ammonium chloride (NH4Cl)23 (Fig. 2f) and bafilomycin A124 (Fig. 2g) strongly reduced FLUAV infection of desialylated MHC II+ HEK-293T cells, suggesting that upon interaction with MHC II, FLUAV follows a similar endocytic pathway as when SA is used as a receptor.

HEK-293T cells allow entry of FLUAV but they do not support efficient FLUAV replication. Therefore, to assess whether MHC II allows replication of FLUAV in desialylated airway epithelial cells, we used A549 cells due to their high transfection efficiency, high permissibility to FLUAV replication, and as a representative of the primary target cells of FLUAV in mammalian hosts. To fully assess the SA independence of MHC II-mediated entry, we suppressed cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS) expression by shRNA silencing. CMAS is an enzyme that catalyzes the activation of Neu5Ac to CMP-Neu5Ac, and its suppression has been shown to abolish FLUAV infection in A549 cells25. Lentivirus-mediated shRNA-CMAS transduction efficiently prevented SA incorporation on the cell surface (Fig. 3a and Supplementary Fig. 7a, b). These shRNA-CMAS cells were non-susceptible to either hVIC/11 or sOH/04 infection, and transfection with a shRNA-resistant CMAS cDNA construct (shRNA-CMAS+CMAS) restored the susceptibility of the cells to FLUAV infection alongside SA presentation on the surface (Supplementary Fig. 7c, d). Cells transduced with a scrambled shRNA control (shRNA-scrambled) showed no differences in SA content and FLUAV replication compared to the parental A549 cells. SLA-DR and HLA-DR expression was consistently observed on 70% of the cells on average (Fig. 3b, c). CMAS repression prevented H3N2 FLUAV replication. However, MHC II expression granted replication in a species-specific manner: HLA-DR expression allowed replication of hVIC/11 in shRNA-CMAS cells (Fig. 3d) while SLA-DR expression enabled replication of sOH/04 to similar levels as the shRNA-CMAS+CMAS control group (Fig. 3e). Interestingly, sOH/04 also replicated in HLA-DR+ cells, although titers at all timepoints were lower compared to infection in SLA-DR+ cells. We then tested whether anti-MHC II antibodies inhibit FLUAV replication in desialylated MHC II+ A549 cells. The anti-HLA-DR antibody strongly reduced hVIC/11 titers in shRNA-CMAS HLA-DR+ cells (Fig. 3f), while the anti-SLA-DR antibody showed a similar effect in sOH/04 titers in shRNA-CMAS SLA-DR+ cells (Fig. 3g). Finally, we evaluated if infection of MHC II+ cells can induce cell death. hVIC/11 replication led to a significant increase in shRNA-CMAS HLA-DR+ cells mortality at 48 hpi (Fig. 3h), similar to the shRNA-scrambled and shRNA-CMAS+CMAS control groups. Conversely, sOH/04 replication resulted in a significant increase in cell death in SLA-DR+ cells, although it also affected HLA-DR+ cells’ viability (Fig. 3i). Altogether, these results show that MHC II can be used as an entry receptor, and MHC II-mediated entry of FLUAV in cells lacking SA leads to productive infection and cell death.

Mutations near the sialic acid receptor binding site of H3N2 FLUAV HA alter species-specificity of MHC II-mediated entry
H2N2 FLUAV has been shown to interact with MHC II via the HA1 domain12. Comparison of hVIC/11 and sOH/04 HA revealed several amino acid differences in the HA1 domain (Supplementary Fig. 8), and no obvious pattern could be detected that could account for differences in their MHC II binding preference. Nonetheless, we have previously shown that adaptation of hVIC/11 to pigs selected for three mutations within the HA RBS: A138S, V186G, and F193Y (H3 numbering), which resulted in increased replication in SA-bearing swine cells18. These mutations spontaneously appeared in different mutant viruses after infection of pigs with hVIC/1118. All these residues are located in the surroundings of the SA-binding pocket, although they do not directly interact with SA (Fig. 4a). Furthermore, the A138S amino acid change significantly increases affinity for PAMs in vivo16. Therefore, we evaluated the ability of mutant hVIC/11-A138S, hVIC/11-V186G, and hVIC/11-F193Y to utilize either HLA-DR or SLA-DR as a receptor in vitro. Infection of desialylated MHC II+ HEK-293T cells revealed a preference for SLA-DR over HLA-DR (Fig. 4b, c). When only MHC II+ cells were considered, a similar trend was observed, with all three mutant viruses using both HLA-DR and SLA-DR as entry receptors, but with a statistically significant higher preference for the swine homologue (Fig. 4d). Quantification of FLUAV-positive cells among total cells (regardless of MHC II expression) supported that infection of desialylated cells is completely abolished even when cells are transfected with an empty plasmid (Fig. 4e). Evaluation of the replication capacity of the viruses in MHC II-transfected, shRNA-CMAS A549 cells, revealed an increase of the viral titers over time in both HLA-DR- and SLA-DR-expressing cells, with no differences observed between the MHC II from both species (Fig. 4f–h). This contrasts with hVIC/11 (Fig. 3d), which failed to efficiently replicate in SLA-DR-expressing cells without SA. Overall, these data demonstrate that the introduction of swine-adapting point mutations near the receptor binding site of hVIC/11 increases the affinity of the virus for SLA-DR. This is consistent with previous reports suggesting that these mutations represent an advantage for the virus in the swine host18. Additionally, we tested whether these mutations could also affect the SA-binding preference of the virus. By performing a solid-phase SA receptor-binding assay, we observed that hVIC/11-A138S and hVIC/11-F193Y exhibited higher affinity for α2,6 SA compared to hVIC/11 (Fig. 5a, b, d), although no changes were seen for α2,3 binding. Contrary, hVIC/11-V186G (Fig. 5c) had a reduced binding to both α2,3 and α2,6 compared to all the mutants, including the parental hVIC/11. Taken together, these results demonstrate that mutations in the proximity of the SA RBS can have a dual effect and impact MHC II recognition and SA binding at the same time.

Methods
Ethics statement
Animal studies described in this report were approved by the Institutional Animal Care and Use Committee at the University of Georgia (protocol A2019 03-031-Y3-A9). 3-week-old healthy cross-bred pigs were obtained from Midwest Research Swine Inc. (Gibbon, MN) and housed in animal biosafety level 2 conditions according to the Guide for the Care and Use of Agricultural Animals in Research and Teaching. At the end of the study, animals were euthanized following the American Veterinary Medical Association (AVMA) guidelines. Sex was not a variable considered in the study, but both sexes were included.

Cells and viruses
Madin-Darby canine kidney (MDCK) were a kind gift from Dr. Robert Webster. Human lung carcinoma (A549) and human embryonic 293T (HEK-293T) cells were purchased from the American Type Culture Collection (ATCC). Cells were maintained in Dulbecco’s Modified Eagle's Medium (DMEM, Sigma-Aldrich, St Louis, MO) supplemented with 2 mM L-glutamine (Sigma-Aldrich, St Louis, MO), 10% fetal bovine serum (FBS, Sigma-Aldrich, St Louis, MO), and 1% antibiotic/antimycotic (Sigma-Aldrich, St Louis, MO). Cells were incubated at 37°C under 5% CO2.
FLUAV viruses in this study were generated using an 8-plasmid reverse genetics system as previously described46,47 using an isogenic backbone. HA sequences were confirmed by Sanger sequencing, and viral stocks were made in MDCK cells in opti-MEM containing 1 μg/mL of tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Worthington Biochemicals, Lakewood, NJ) at 37°C. Viral titers were determined by TCID50 using the Reed and Muench method48.

Generation of A549 cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS) deficient cells
CMAS expression was silenced by using a shRNA lentiviral vector. Inverted, self-complementary hairpin DNA oligos targeting the CMAS mRNA (shRNA-CMAS: 5’-AAGAGTGCTTGAAGAGAGTGG-3’) were cloned into a pLKO.1 plasmid (kindly donated by David Root, Addgene plasmid #10878) under the control of the human RNA polymerase III U6 promoter49. To generate lentiviruses encoding CMAS shRNA, or the scrambled shRNA negative control (kindly donated by David Sabatini50, addgene plasmid # 1864), HEK-293T cells were co-transfected with pCMVdr8.2dvpr, pCMV-VSV-G, and the corresponding pLKO.1 shRNA plasmid. Supernatants were collected at 24, 48, and 72 h post-transfection. Supernatants were pooled, and cell debris was discarded by centrifugation at 500 x g for 5 min and further filtration through a 0.45 μm filter. Lentiviruses were concentrated using the Lenti-X concentrator (Takara Bio, Mountain View, CA) for 6 h and resuspended in 1/100th of the original volume according to the manufacturer’s instructions. A549 cells were transduced with pLKO.1-shRNA-CMAS-puro- or pLKO.1-scrambled-shRNA-puro-encoding lentiviruses with 8 μg/mL polybrene (Sigma-Aldrich, St Louis, MO). At 48 h post-transduction, cells were incubated with media containing 1 μg/mL puromycin (Thermo Fisher Scientific, Waltham, MA). Successful CMAS silencing was confirmed by measuring SA levels on the cell surface using flow cytometry and immunofluorescence as described below. Flow cytometry data was analyzed with FlowJo v10.10.0 and confocal images with ImageJ v1.54 p and Fiji v2.16.0.

Porcine alveolar macrophage isolation
Swine alveolar macrophages were collected and isolated as previously described51,52 with minor modifications. Three-week-old crossbred pigs were obtained from Midwest Research Swine Inc. (Glencoe, MN). Animals were housed under biosafety level 2 conditions at the University of Georgia. After a 7-days acclimatation period, animals were confirmed negative for anti-FLUAV antibodies was performed as previously described16,53. These cells exhibited a CD172a pos CD163 high MHC II pos phenotype.

Tissue immunofluorescence
Right cranial lobe samples were collected from infected (sOH/04) and non-infected pigs in neutral-buffered formalin and paraffin embedded in a previous study16. For SLA-DR and HA detection, samples were deparaffinized and stained as previously described54 with minor modifications. Briefly, lung sections were deparaffinized and rehydrated, followed by an antigen retrieval step in citrate buffer (10 mM sodium citrate, pH 6.0) for 40 min. Samples were permeabilized with 0.3% Triton-X100 (Sigma-Aldrich, St Louis, MO) and blocked with 5% Bovine Serum Albumin (BSA, Sigma-Aldrich, St Louis, MO) for 1 h. FLUAV was detected using an anti-Multi-Hemagglutinin (H3N2) polyclonal antibody (eEnzyme, IA-PAN4-0100, Gaithersburg, MD), while an anti-pig SLA class II DR clone 2E9/13 (Bio-Rad, MCA2314GA, Hercules, CA) for MHC II detection was used. Samples were then incubated with a secondary 594-conjugated anti-rabbit antibody in a 1:1000 dilution for HA detection and an Alexa 488-conjugated anti-mice antibody in a 1:1000 dilution for SLA-DR. After a 1-h incubation, tissue samples were stained with 0.5 μg/mL 4’,6-diamine-2-phenylindole (DAPI, Sigma-Aldrich, St Louis, MO) for 15 min. Finally, samples were mounted on glass slides with mounting media (Vector Laboratories, Newark, CA) and imaged using a ZEISS LSM900 confocal microscope. Images were analyzed with ImageJ v1.54 p and Fiji v2.16.0.

Sialic acid removal of PAMs and HEK-293T cells
α2,3 and α2,6-SA were removed from the cell surface by incubating cells with 100 mU/mL of neuraminidase from Clostridium perfringens (Sigma-Aldrich, St Louis, MO) for 24 h prior to infection. After infection, fresh neuraminidase was added, and new neuraminidase was added every 24 h post-infection. Sialic acid hydrolysis was confirmed by immunofluorescence and flow cytometry using fluorescein-conjugated SNA (Vector Laboratories, Newark, CA) and biotinylated MALII (Vector Laboratories, Newark, CA). Flow cytometry data were analyzed with FlowJo v10.10.0.

Viral growth kinetics
Isolated PAMs were seeded in a 12-well plate at a density of 1.5 × 10^5 cells/well. Similarly, A549 cells were seeded in a 6-well plate at a density of 5 × 10^5 cells/well. All cells were incubated at 37°C overnight and then infected at an MOI of 0.01 for 1 h at 37°C. After infection, plates were washed three times with PBS and cells were supplemented with Opti-MEM (Thermo Fisher Scientific, Waltham, MA) containing 0.1 μg/mL of TPCK-treated trypsin. Supernatant aliquots were collected at 0, 12, 24, 48, and 72 h post-infection and kept at –80°C until processing. Viral RNA was extracted using the MagMax-96 AI/ND viral RNA (Thermo Fisher Scientific, Waltham, MA) isolation kit according to manufacturer’s instructions. Purified RNA was used as template for RT-qPCR using the Quantabio qScript XLT One-Step RT-qPCR ToughMix kit (Quantabio, Beverly, MA) and specific primers targeting the M segment of the virus (Supplementary Table 1). FLUAV titers were determined by comparison to a TCID50 eq standard curve of an exact virus match of known titer previously determined using the Reed and Muench method48.

HLA-DR and SLA-DR antibody blocking
PAMs were seeded in 12-well plates at a density of 1.5 × 10^5 cells/well while A549 cells were plated in a 6-well plate at a density of 5 × 10^5 cells/well. Plates were incubated overnight at 37°C. The next day, cells were incubated for 1 h at 4°C with an anti-SLA-DR (Bio-Rad, Hercules, CA) or an anti-HLA-DR (Thermo Fisher Scientific, Waltham, MA) antibody at concentrations ranging from 1 to 10 μg/mL or an anti-GFP antibody as a control (Sigma-Aldrich, St Louis, MO). After incubation, cells were infected at an MOI of 0.1 for 1 h at 37°C in the presence of the indicated antibody concentrations. Finally, cells were washed three times with PBS and supplemented with Opti-MEM containing 0.1 μg/mL of TPCK-treated trypsin and the desired antibody concentrations. Samples were collected at 48 hpi for further virus titration.

Plasmids
HLA-DRA (GenBank accession number: NM_019111.5), HLA-DRB1 (GenBank accession number: NM_002124.4), SLA-DRA (GenBank accession number: MF498819.1), and SLA-DRB1 (GenBank accession number: MF498811.1) gene fragments were synthetized (Twist Bioscience, San Francisco, CA) and amplified by PCR to introduce EcoRI and BglII restriction sites using the primers listed in Supplementary Table 1. pCAGGS plasmid and MHC II fragments were digested using EcoRI and BglII restriction enzymes (New England Biolabs, Ipswich, MA) according to the manufacturer’s instructions for 1 h at 37°C for further ligation and transformation. Plasmid sequences were confirmed by whole plasmid sequencing.

MHC II expression in A549 and HEK-293T cells
Cells were seeded at a density of 8 × 10^5 cells/well in 6-well plates and were incubated at 37°C until a 70% confluence was reached. MHC II subunits were transfected using equimolar quantities of either HLA-DRA and HLA-DRB1 or SLA-DRA and SLA-DRB1 and TransIT LT1 (Mirus Bio, Madison, WI) in a 1:2 ratio DNA: TransIT. HLA-DR and SLA-DR complexes expression was assessed by flow cytometry and immunofluorescence as described below. At 24 h post-transfection, cells were desialylated as described above, and non-sialylated cells were infected at 48 h post-transfection. Flow cytometry data were analyzed with FlowJo v10.10.0 and confocal images with ImageJ v1.54 p and Fiji v2.16.0.

MHC II-mediated entry assessment
HEK-293T cells were seeded and transfected as described above. At 24 h post-transfection, HEK-293T cells were desialylated with 100 mU/mL of neuraminidase from Clostridium perfringens for 24 h at 37°C. At 48 h post-transfection, cells were infected at an MOI of 0.1 for 1 h at 37°C. After incubation, the virus-containing supernatant was discarded, and fresh media supplemented with 0.1 μg/mL TPCK-treated trypsin and 100 mU/mL of neuraminidase was added. Neuraminidase was not included in the non-desialylated control groups. Finally, infections were incubated for 24 h at 37°C for further processing.

Flow cytometry analysis
Twenty-four hours after infection, supernatants from HEK-293T-infected cells were collected, and cells were trypsinized and centrifuged at 500 x g for 5 min. Cells were resuspended in PBS supplemented with 5% FBS and stained with the LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Cells were then fixed with 4% paraformaldehyde for 15 min at room temperature, followed by a 15-min permeabilization with 0.3% Triton-X (Sigma-Aldrich, St Louis, MO). Afterwards, cells were washed three times and incubated for 1 h with the following primary antibodies: Alexa 647-conjugated anti-SLA-DR clone 2E9/13 (Bio-Rad, Hercules, CA), anti-HLA-DR clone LN3 (Thermo Fisher Scientific, Waltham, MA) at a 1:200 dilution in PBS, and an anti-Multi-Hemagglutinin (H3N2) polyclonal antibody (eEnzyme, IA-PAN4-0100, Gaithersburg, MD) at a 1:500 dilution. Following incubation, cells were washed three times with PBS. For simultaneous detection of FLUAV and MHC II, samples were incubated for 1 h with an Alexa 488-conjugated anti-human secondary antibody and an APC-conjugated anti-mice secondary antibody, respectively (Thermo Fisher Scientific, Waltham, MA), both in a 1:1000 dilution. Finally, cells were washed three times with PBS and analyzed using a NovoCyte Quanteon flow cytometer system (Agilent, Santa Clara, CA) using the gating strategy exemplified in Supplementary Fig. 9. Data were analyzed using FlowJo version 10.10.0 (FlowJo, Ashland, OR).

Immunofluorescence staining
HEK-293T and A549 cells were plated in poly-D-Lysine-coated glass slides and incubated at 37°C until an 80% confluence was reached. Cells were transfected and desialylated as described above. Cells were infected at an MOI of 0.01 and incubated for 24 h at 37°C as described above. After infection, cells were fixed with 4% formaldehyde for 30 min at room temperature, followed by a 10-min permeabilization with 0.3% Triton-X100 and a 1-h blocking step using 3% bovine serum albumin (BSA, Thermo Fisher Scientific, Waltham, MA) and 0.1% Triton-X100 in PBS. HLA-DR, SLA-DR, and FLUAV were stained using the specific antibodies described above in a 1:200 dilution for 1 h at room temperature in 3% BSA and 0.1% Triton-X100 in PBS. After incubation, samples were washed three times with PBS and incubated with the secondary antibodies described above in a 1:1000 dilution with 0.5 μg/mL 4’,6-diamine-2-phenylindole (DAPI, Sigma-Aldrich, St Louis, MO), 3% BSA, and 0.1% Triton-X100 in PBS for 1 h. Finally, cells were washed 5 times with PBS and mounted on glass slides using mounting media. For SA staining, samples were incubated with either biotin-conjugated or fluorescein-labeled SNA (Vector Laboratories, Newark, CA) and biotin-conjugated MALII (Vector Laboratories, Newark, CA) lectins in a 1:250 dilution in PBS for 30 min prior to permeabilization. After incubation with lectins, cells were incubated for 30 min with a secondary Alexa 594-conjugated streptavidin in a 1:1000 dilution. Samples were imaged using a ZEISS LSM900 confocal microscope. Images were analyzed with ImageJ v1.54 p and Fiji v2.16.0.

FLUAV entry blockage and cell viability assay
HEK-293T cells were plated and transfected as described above. 1 h before infection, cells were incubated with 20 mM of NH4Cl (Sigma-Aldrich, St Louis, MO) or 10 nM Bafilomycin A1 (Cell Signaling Technologies, Danvers, MA). After incubation, cells were infected at an MOI of 0.1 as described above and kept at 37°C for 24 h in the presence of the desired inhibitor. At 24 hpi, cells were washed three times with PBS, and total cellular RNA was extracted using the MagMax-96 AI/ND RNA (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. RNA samples were normalized to 1 μg, and FLUAV M segment copy number was determined by RT-qPCR using the Quantabio qScript XLT One-Step RT-qPCR ToughMix kit (Quantabio, Beverly, MA) and a standard curve of an M-cloned segment plasmid of known concentration.
Cellular viability was assessed using the CellTiter Glo Cell Viability kit (Promega, Madison, WI) according to the manufacturer’s instructions.

MHC II-mediated replication in A549 cells
Parental, shRNA-scrambled, and shRNA-CMAS A549 cells were seeded and transfected as described above. At 48 h post-transfection, cells were infected at an MOI of 0.01 for 1 h at 37°C. After incubation, the supernatant was discarded and replaced with media containing 0.1 μg/mL TPCK-treated trypsin. Time points were collected at 0, 12, 24, 48, and 72 h post-infection, and viral titers were determined by RT-qPCR using a standard curve generated from 10-fold serial dilutions of a virus stock with a known TCID50 titer.

Solid-phase binding assay
Virus affinity for α2,3 and α2,6 SA was assessed as previously described55. Briefly, viruses were purified by ultracentrifugation at 140,000 x g for 3 h using a 20% sucrose cushion. Pelleted viruses were resuspended in TNE buffer (0.01 M Tris, 0.001 M EDTA, 0.1 M NaCl, pH 7.2). Samples were normalized to 128 HAU and adsorbed in fetuin-coated plates at 4°C overnight. Unbound virus was washed using 0.02% Tween-80 in PBS (washing buffer), and plates were blocked for 2 h with desialylated BSA (BSA-NA). After blocking, plates were washed three times with washing buffer and incubated with different concentrations of Neu5Acα2-3Galβ1-4GlcNAcβ-PAA-biotin (α2,3 SA, Glycotech, Gaithersburg, MD) or Neu5Acα2-6Galβ1-4GlcNAcβ-PAA-biotin (α2,6 SA, Glycotech, Gaithersburg, MD) in 0.02% Tween-80, 0.1% BSA-NA, and 2 μM oseltamivir in PBS (reaction buffer). After a 1-h incubation, plates were washed five times with washing buffer and incubated with HRP-conjugated streptavidin (Thermo Fisher Scientific, Waltham, MA) in a 1:1000 dilution for 1 h at 4°C. Finally, plates were washed five times with washing buffer and incubated with TMB (Thermo Fisher Scientific, Waltham, MA) for 10 min. Reaction was stopped using a stop solution (Thermo Fisher Scientific, Waltham, MA), and absorbance was measured at 450 nm using a Synergy HTX Multi-Mode Microplate Reader Agilent BioTek, Santa Clara, CA).

Statistical analysis
Statistical analyses were performed using GraphPad Prism 10 software. Data is presented as the mean ± standard error of the mean of at least three independent experiments unless stated otherwise in the figure legend. Each data point represents the average of three technical replicates for a total of three biological replicates per experiment. P values were obtained by ordinary two-way ANOVA with Tukey’s multiple comparison test.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
All source data relevant to this study are available within the manuscript and have been provided as a source data file and supplementary information. Additionally, the following databases were used in this study: Protein Data Bank (PDB, https://www.rcsb.org/) and National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/) Sourced data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Comments on “MHC class II is a functional receptor for H3N2 influenza A viruses and mediates host-specificity” (NCOMMS-25-81023-T)\nI would like to thank the authors for addressing my comments, especially the new experiments that suppressed the expression of CMAS in A549 cells. I have the following comments/ questions for consideration.\n1. The rationale of selecting the two H3N2 viruses in this study was due differences previously observed between hVIC/11 and sOH/04 in infecting porcine alveolar macrophages (PAM) (PMID: 38377132). In the same study, the authors also reported that the HA-A138S change in hVIC/11 increased infectivity in PAM. The authors should report the genetic differences between the HA proteins of hVIC/11 and sOH/04, which would be important to explain why the A138S, V186G, and F193Y changes were later investigated in the manuscript. In line with this comment, the authors should also discuss why A138S showed increased infectivity of PAM (PMID: 38377132) but did not appear to preferentially use SLA-DR over HLA-DR as the sOH/04 virus. If A138S, V186G, and F193Y changes were the only amino acid differences between the two HA, the authors should consider changing all three in hVIC/11 to see if they switch the preference use HLA-DR to SLA-DR. This result will further support the direct interaction between HA and MHC II, as current data is limited in supporting this point (eg. single mutations showed similar result as hVIC/11).\n2. C. perfringens neuraminidase was used to cleave sialic acids followed by lectin staining to confirm removal of sialic acid. Pig cells express both NeuAc and NeuGc and it would be helpful if the authors can report C. perfringens substrate specificity (eg. efficiency in removing NeuGc and Kdn), if there are any previous literatures. In addition, it was noted that SNA and MAL II were used for detection of sialic acid, it is important to note that MAL II binds to alpha2,3-linked O-glycans, while MAL I binds to alpha2,3-linked LacNAc, which was not used in this study. It is also not clear if these lectins will bind to NeuGc as efficiently. Due to these limitations, I will suggest the authors to be more conservative when describing the results after C. perfringens neuraminidase treatment. For example, Lines 128-130, …transfected cells were treated with exogenous neuraminidase and neither a2,6-SA nor a2,3-SA on the cell surface was detected after a 24-hour neuraminidase treatment.\n3. The authors should discuss if binding to MHC II is common among H3N2 viruses or if this is a unique observation of the selected viruses.\nSpecific comments:\n1. Supplementary Fig. 4 title. Please change deacetylated cells to de-sialylated cells.\n2. Line 219. Fig. 3c should this be Fig. 3d?\n3. The title of the manuscript may be modified to match the results better. Currently only 2 H3N2 viruses were characterized, and extrapolating the results to infer H3N2 host-specificity may be a bit overstated.", "reviewer_2": "While the authors have clearly shown that the swine adapted H3N2 (sOH/04) mediates infection through SLA (swine MHCII), the biological relevance has not been established.\nAlthough the authors nicely addressed several of the specific comments of this reviewer’s evaluation of the initial submission, the most substantive comment in the summary paragraph above was not directly addressed. In particular, since the relevance of virus infection through MHC II cannot be established in vitro unless sialic acids are first removed (by neuramindase or ablation of CMAS), what is the biological relevance of this alternative pathway for infection in swine? It is generally considered that the influenza virus infection is initiated in epithelial cells lining the airway. If so, what cells would express MHC II but be deficient in sialic acid?", "reviewer_3": "1. Line 58-60: Could the authors clarify or modify the statement to reflect the previous findings referenced here (Karakus et al., 2019)? Does this refer to Fig 3e of Karakus et al., where it appears that although there is H17 entry in cells expressing HLA-DR, there is also entry (albeit reduced) into SLA-DR, E. fuscus DR, and M. lucifugus DR?\n2. Line 93-95/Figure 1a: Was hVIC/11 not expected to co-localize with SLA-DR? If so, could this be included to show species specificity and act as a negative control along with the mock?\n3. Line 105: Did the authors consider collecting a quantitative measure of sialic acid abundance via flow cytometry, for example? While there is a visual difference based on the IF results (Supplemental Fig 1b), it weakens the later point that this is a sialic acid independent mechanism, as it is not clear how much sialic acid remains in the 100mU condition.\n4. Line 111: Where the authors state that inhibition “seemed proportional,” could they provide a more quantitative description of that result?\n5. Line 116 (and line 729): What is the control antibody? Please provide more information.\n6. Line 133-137: (Supplemental Figure 3b & Figure 2c): It appears there is an increase in the percentage of infected cells with sOH/04 in the de-acetylated+SLA-DR (2c) condition versus the wildtype acetylated cells (Supplemental Figure 3b) (28.8% versus 3.38%). Does this suggest that sOH/04 prefers SLA-DR over SA as a receptor or was SLA-DR highly abundant on the surface as compared to sialic acid? If there is data showing SLA-DR abundance, this should be provided in Supplemental Figure 3b. This could be an interesting finding for the authors to discuss further.\n7. Line 153 and figure 2f the labelling in this figure is confusing. Does vehicle refer to untreated cells and then the HLA-DR and SLA-DR are both NH4Cl treated? If so, is the vehicle the control data for the HLA or for the SLA?\n8. Line 165-167: Could SNA or MAL have been added in other assays to block remaining SA?\n9. Line 166: Please explain the use of the peanut lectin more clearly in these blocking experiments- how does this lectin block remaining/ exposed sialic acid? PNA binds to a core 1 O-glycan (not sialylated) and does not bind if there is anything on the galactose. See figure 5 of this paper (https://pubs.acs.org/doi/epdf/10.1021/acschembio.1c00689?ref=article_openPDF). Correct in the materials and methods that it binds to “exposed B1-3 Nacetylgalactosamine” to “GalB1-3N-acetylgalactosamine” or “Tn antigen”.\n10. Line 190-191 Make clearer from the outset that the three mutations that are investigated were each seen individually in different mutants and did not occur in the same genome- otherwise it is a bit confusing to begin with why they are considered individually.\n11. Line 193/Fig 4a: Could the authors highlight the full SA binding pocket for reference?\n12. Line 213-215/Fig 5: Have the authors tried removing these mutations from sOH/04 to yield a null effect?\n13. Line 230: As sialic acid is so pervasive on the cell surface, the authors were not able to remove it completely (Line 105). How do the authors know that this is a sialic independent mechanism rather than a mechanism which can be used in conjunction with low concentrations of sialic acid?\nThroughout please refine various wordings:\n14. “Sialic acid conformation” should be corrected to “sialic acid linkage configuration” in the abstract. Conformation refers to arrangement of atoms in a molecule around a bond - which is not the case for 2,3 vs 2,6 as you need to break and reform a bond.\n15. Replace deacetylated with “desialylated” - you can deacetylate Neu5Ac (the acetyl group is on C5), but that’s not what neuraminidase does, you’d need an esterase to deacetylate.\nPlease double check this is correct throughout (for example supplemental figure 4 and figure 1 legend).\nNew additional comments:\n1. Although the authors mention in response to Reviewer 1 that A/California/04/2009(H1N1) is a suitable negative control for the specificity of their assays, could the authors please explain why a positive control was not included in their experiments? If this is not feasible, I would encourage the authors to elaborate on that in their response.\n2. Line 100 in the revised manuscript refers to PAMS that were collected from naïve pigs in a previous study. Do the authors really mean they used cells that had been previously collected and perhaps frozen or do they mean collected as in a previous study? Please clarify.\n3. Line 179: the words used ‘observed around 70%’ are confusing please rephrase more clearly- what is around 70%- were 70% of the cells expressing SLA-DR?\n4. Line 246 the authors use the term ‘on line,’ would ‘in line with’ be better?\n5. Line 259 the authors use the word ‘remanent’, ‘remaining’ or ‘residual’ would be clearer.\n6. Throughout the manuscript the authors refer to both replication and entry interchangeably. It would be useful for the overall clarity of the manuscript if the authors could please define these terms and be more precise with their use. For example, clearly define how each assay was read out in the text (such as the TCID50 equivalent standard curve in Figure 3) and explain how this relates to a productive infection. In Figures 2 and 3, with HA-positive or vRNA readouts, these assays seem to provide evidence for entry and transcription, but not necessarily productive replication and infectious progeny, as in Figure 1."}, "author_response": "Point-by-point response to reviewers - NCOMMS-25-81023-T\nWe appreciate the reviewers’ time and effort spent providing feedback and insightful\ncomments to help improve our manuscript “MHC class II functions as a host-specific\nentry receptor for representative human and swine H3N2 Influenzas viruses”. We have\naddressed the reviewers’ comments and incorporated changes to the manuscript.\nPlease see below a point-by-point response to the reviewers’ comments and concerns.\nLine numbers refer to the revised manuscript file with tracked changes.\nReviewer comments:\nReviewer #1\nI would like to thank the authors for addressing my comments, especially the new\nexperiments that suppressed the expression of CMAS in A549 cells. I have the following\ncomments/ questions for consideration.\n1. The rationale of selecting the two H3N2 viruses in this study was due differences\npreviously observed between hVIC/11 and sOH/04 in infecting porcine alveolar\nmacrophages (PAM) (PMID: 38377132). In the same study, the authors also reported\nthat the HA-A138S change in hVIC/11 increased infectivity in PAM. The authors should\nreport the genetic differences between the HA proteins of hVIC/11 and sOH/04, which\nwould be important to explain why the A138S, V186G, and F193Y changes were later\ninvestigated in the manuscript. In line with this comment, the authors should also\ndiscuss why A138S showed increased infectivity of PAM (PMID: 38377132) but did not\nappear to preferentially use SLA-DR over HLA-DR as the sOH/04 virus. If A138S,\nV186G, and F193Y changes were the only amino acid differences between the two HA,\nthe authors should consider changing all three in hVIC/11 to see if they switch the\npreference use HLA-DR to SLA-DR. This result will further support the direct interaction\nbetween HA and MHC II, as current data is limited in supporting this point (eg. single\nmutations showed similar result as hVIC/11).\nR: Thank you for pointing this out. As per your request, we have incorporated a new\nSupplementary Figure 8 of a multiple sequence alignment between hVIC/11 and\nsOH/04. Multiple amino acids differences can be observed in the HA1 domain which\nhas been shown to mediate binding to MHCII 1. Therefore, we cannot draft any\nconclusions on what residues might be contributing to MHCII specificity. However, we\nhave used this analysis to introduce mutations studied. This is now presented in lines\n223-227: “H2N2 FLUAV has been shown to interact with MHCII via the HA1 domain.\nComparison of hVIC/11 and sOH/04 HA revealed several amino acid differences in the\nHA1 domain, and no obvious pattern could be detected that could account for\ndifferences in their MHCII binding preference. Nonetheless, we have previously showed\nthat adaptation of hVIC/11 to pigs selected for three mutations within the HA RBS:\nA138S, V186G, and F193Y (H3 numbering), which resulted in increased replication in\nSA-bearing swine cells 2.”\nAs you correctly noted, A138S does not show a clear preference for either MHC II\nhomolog. However, it is able to replicate in presence of SLA, which hVIC/11 poorly\ndoes. This aligns with our previous observations showing that hVIC/11-A138S infects\nand replicates in a swine macrophage cell line, albeit less efficiently than sOH/04, while\nhVIC/11 fails to replicate in these cells altogether 3. Similarly, in vivo, sOH/04 infects up\nto ~70% of macrophages in the lungs, whereas hVIC/11-A138S reaches no more than\n~40%, and hVIC/11 is not detected in PAMs from challenged animals 3. Thus, the\nreduced ability of the A138S mutant to infect and replicate in macrophages compared to\nsOH/04 is fully consistent with our SLA utilization data presented here. This has been\nadded to the discussion section lines 377-380: “In a previous report we found that the\nA138S mutation in a hVIC/11 HA increased affinity for PAMs in vivo but did not reach\nthe same level of affinity as the swine-adapted sOH/04 3. This effect is consistent with\nthe dual usage of SLA-DR by hVIC/11-A138S and the inability of hVIC/11 to efficiently\nreplicate in SLA-DR-positive cells.”\n2. C. perfringens neuraminidase was used to cleave sialic acids followed by lectin\nstaining to confirm removal of sialic acid. Pig cells express both NeuAc and NeuGc and\nit would be helpful if the authors can report C. perfringens substrate specificity (eg.\nefficiency in removing NeuGc and Kdn), if there are any previous literatures. In addition,\nit was noted that SNA and MAL II were used for detection of sialic acid, it is important to\nnote that MAL II binds to alpha2,3-linked O-glycans, while MAL I binds to alpha2,3-\nlinked LacNAc, which was not used in this study. It is also not clear if these lectins will\nbind to NeuGc as efficiently. Due to these limitations, I will suggest the authors to be\nmore conservative when describing the results after C. perfringens neuraminidase\ntreatment. For example, Lines 128-130, …transfected cells were treated with\nexogenous neuraminidase and neither a2,6-SA nor a2,3-SA on the cell surface was\ndetected after a 24-hour neuraminidase treatment.\nR: Thank you for bringing this up. The neuraminidase used in this study correspond to\nthe NanI neuraminidase from C. perfringens. This enzyme can cleave both 2,3- and\n2,6-SA 4, and it has been shown to also cleave Neu5Gc albeit less efficiently\ncompared to Neu5Ac 5. Additionally, the SNA lectin can bind almost equally to either\n2,6-Neu5Ac or 2,6-Neu5Gc 6,7. However, due to the differences in affinity, we agree\nthat we should be more conservative in our conclusions. We have incorporated this\nreferences in the main text an lines 106-108 now read: “Two methods were evaluated to\ndeplete SA on the cell surface: chemical oxidation and enzymatic SA removal with the\nNanI neuraminidase from Clostridium perfringens, known to cleave both 2,3- and 2,6-\nNeu5Ac 4 and Neu5Gc 5”\nWe agree that we should be more conservative in our statements and therefore have\nmodified accordingly in sections MHCII is needed for the infection of porcine\nalveolar macrophages (PAMs) by swine-adapted H3N2 FLUAV in the absence of\nSAs and Transient expression of MHCII in desialylated cells enables FLUAV entry\nand replication.\n3. The authors should discuss if binding to MHC II is common among H3N2 viruses or if\nthis is a unique observation of the selected viruses.\nR: Thank you for pointing this out, because of the limited number of viruses tested here,\nwe would like to refrain from extrapolating this finding to all H3N2 viruses. We have\nadded sentence in discussion between lines 304-306: “However, because only a limited\nnumber of viruses were tested, it remains unclear whether other H3N2 viruses can\nutilize MHCII, especially since MHCII binding can vary within a subtype (e.g. H2N2\nviruses)”\nSpecific comments:\n1. Supplementary Fig. 4 title. Please change deacetylated cells to de-sialylated cells.\nR: Thank you for pointing this out. We have modified the figure title accordingly.\n2. Line 219. Fig. 3c should this be Fig. 3d?\nR: Thank you for pointing this out. We have modified the text accordingly.\n3. The title of the manuscript may be modified to match the results better. Currently only\nspecificity may be a bit overstated.\nR: We appreciate the reviewer’s comments. We agree with your suggestion and have\nmodified the title to better reflect our findings. The manuscript title now reads: “MHC\nclass II functions as a host-specific entry receptor for representative human and swine\nH3N2 influenza A viruses”.\nReviewer #2\n1. While the authors have clearly shown that the swine adapted H3N2 (sOH/04)\nmediates infection through SLA (swine MHCII), the biological relevance has not been\nestablished.\nAlthough the authors nicely addressed several of the specific comments of this\nreviewer’s evaluation of the initial submission, the most substantive comment in the\nsummary paragraph above was not directly addressed. In particular, since the\nrelevance of virus infection through MHC II cannot be established in vitro unless sialic\nacids are first removed (by neuramindase or ablation of CMAS), what is the biological\nrelevance of this alternative pathway for infection in swine? It is generally considered\nthat the influenza virus infection is initiated in epithelial cells lining the airway. If so, what\ncells would express MHC II but be deficient in sialic acid?\nR: We thank the reviewer for raising this important point. We agree that sialic acids\nremain the primary receptors for influenza A viruses and that a complete absence of\nsialylated glycans is unlikely in the swine respiratory tract. Our interpretation is not that\nMHCII replaces sialic acid but rather that it can supplement or enhance infectivity under\nspecific biological conditions. In other words, the alternative pathway we describe does\nnot require the absence of sialic acid.\nWe propose that MHCII can facilitate infection when glycan-mediated binding is\nsuboptimal, a scenario likely to occur during adaptation of a human-origin virus to the\nswine host. Glycan array experiments with hVIC/11 HA and its A138S, V186G, F193Y\nvariants show preferential binding to extended human-like tri-LacNAc α2,6 structures 8,\nwhich are uncommon in the swine respiratory tract 9. Under these conditions, HA-glycan\ninteractions are inefficient, and the viruses cannot efficiently engage swine-specific\nglycans due to their topology rather than their absence. In this context, engagement of\nMHCII may provide an additional entry mechanism, effectively rescuing infectivity and\nsupporting early rounds of replication by expanding the repertoire of receptors available,\nthereby increasing the probability of productive infection. Similarly, sOH/04 can utilize\nHLA-DR, suggesting that the same mechanism may apply in the event of reverse\nzoonosis.\nImportantly, sOH/04 efficiently uses SLA-DR and replicates in swine alveolar\nmacrophages3, leading to apoptosis and depletion of these cells in vivo. SLA-DR-\nmediated infection may contribute to the macrophage disappearance reaction observed\nduring FLUAV infection 3 by specifically targeting MHCII-positive cells, potentially\ndelaying viral clearance and reducing immune defense 10,11. In this context, MHCII\nrepresents an additional mechanism for infection of this cell type specifically. Although\nthe swine macrophage glycome has not been characterized, human macrophages are\ndecorated with N-glycans, which make up approximately 40% of all glycans, of which\naround 60% are paucimannosidic and oligomannosidic, which are not FLUAV receptors,\nand only 7% are 2,6-SA-capped 12. These glycans appear branched but shorter than\nthose in lung epithelial cells12,13, which predominantly feature elongated, multi-LacNAc\nN-glycans 13. If swine PAMs share similar glycan features, the use of MHCII as an\nalternative receptor becomes even more relevant for infection of these cells. This is\npartially supported by Figure 2e in which sOH/04 was able to infect a higher proportion\nof cells when only SLA-DR was present compared to when sialic acid was being used\nas a receptor, most likely due to the presence of glycans not supporting sOH/04 efficient\nbinding. Moreover, it has been shown that H2N2 viruses can infect non-professional\nantigen presenting epithelial cells via MHCII despite they being able to bind and\nrecognize sialic acid as an entry receptor 1. Although these cells have not been reported\nin the swine respiratory tract it is very likely that they exist given that they have been\nfound in the gut.\nIn summary, while sialic acids remain the primary entry receptors for influenza A\nviruses, MHCII provides a complementary pathway that can enhance infectivity when\nHA-glycan interactions are suboptimal, particularly in macrophages with limited or short\nsialylated glycans that are not recognized by H3 viruses.\nWe have incorporated this discussion in lines 306-320: “Our data points that sialic acids\nremain the primary receptors for these viruses. Since complete absence of sialylated\nglycans is unlikely in the swine respiratory tract, MHCII could favor infectivity when\nglycan-mediated binding is suboptimal, such as during adaptation of human-origin\nviruses to swine hosts. Glycan array experiments with hVIC/11 HA and its A138S,\nV186G, F193Y variants show preferential binding to extended human-like tri-LacNAc\nα2,6 structures 8, which are uncommon in swine 9, making HA-glycan interactions\ninefficient. In this context, engagement of MHCII provides a selective alternative entry\nroute, rescuing infectivity and supporting early rounds of replication. This is particularly\nrelevant in macrophages, which express robust levels of MHCII and have a glycan\nlandscape with fewer of both elongated and α2,6-sialylated glycans to support HA\nbinding 12.This mechanism is consistent with replication of hVIC/11 A138S and sOH/04\nin swine alveolar macrophages 3, suggesting that MHCII serves as an additional entry\nfactor when glycan-HA compatibility is limited, enabling selective infection of MHCII-\npositive cells.” And lines 307-311: “This is supported by enhanced infection of sOH/04\nin human-origin cells expressing SLA-DR compared to the same cells containing sialic\nacid only (Figure 2e), although the virus did not reach higher levels of replication by\nusing SLA-DR compared to sialic acid (Figure 3e).”\nReviewer #3\n1. Could the authors please provide MFI values alongside Supplementary Fig 3 a &\nb? Additionally, could the authors please elaborate on the high autofluorescence\nin Fig 3a versus Fig 3b? I would also suggest a figure defining the gating strategy\nused here and any other stains including cell viability dyes (please clarify if such\ndyes were used).\nR: As per your request, we have added a new Supplementary Figure 3d and\nSupplementary Figure 7b comparing the MFI values of each sample. The sialic\nacid gating strategy has been added to Supplementary Figure 9 as panel b. The\nsettings used for this experiment may have induced higher levels of\nautofluorescence; however, sialic acid–positive cells exhibit, on average,\napproximately 1000-fold higher signal compared to sialic acid–negative cells,\nallowing for efficient discrimination between populations.\n2. “Line 133-137: (Supplemental Figure 3b & Figure 2c): It appears there is an\nincrease in the percentage of infected cells with sOH/04 in the de-\nacetylated+SLA-DR (2c) condition versus the wildtype acetylated cells\n(Supplemental Figure 3b) (28.8% versus 3.38%). Does this suggest that sOH/04\nprefers SLA-DR over SA as a receptor or was SLA-DR highly abundant on the\nsurface as compared to sialic acid? If there is data showing SLA-DR abundance,\nthis should be provided in Supplemental Figure 3b. This could be an interesting\nfinding for the authors to discuss further.\nR: As you pointed out, we visually observed this effect. However, upon further\nanalysis by flow cytometry we were not able to observe a preference for SLA-DR\nover sialic acid. Therefore, we refrain from drawing conclusions regarding the\nreceptor preference of this specific virus.”\nCould this be added elaborated on in the text?\nR: As per your request, we have incorporated the following changes in the text:\na. Lines 161-1163: “Notably, sOH/04 infected a higher percentage of cells\nwhen SLA-DR was the only available receptor compared to conditions\nwhere only sialic acid was present (Fig. 2e, p = 0.02).”\nb. Lines 307-311: “This is supported by enhanced infection of sOH/04 in\nhuman-origin cells expressing SLA-DR compared to the same cells\ncontaining sialic acid only (Figure 2e), although the virus did not reach\nhigher levels of replication by using SLA-DR compared to sialic acid\n(Figure 3e).”\n3. “Line 230: As sialic acid is so pervasive on the cell surface, the authors were not\nable to remove it completely (Line 105). How do the authors know that this is a\nsialic independent mechanism rather than a mechanism which can be used in\nconjunction with low concentrations of sialic acid?\nR: We appreciate the reviewer’s comment. As pointed out, we were unable to\ncompletely remove sialic acid from the PAMs’ surface. Nonetheless, the\nneuraminidase treatment coupled with an anti-MHCII antibody almost completely\nblocked FLUAV infection strongly suggesting that MHCII is indeed being use as a\nreceptor. This was further confirmed with our data using HEK-293T cells, and\nespecially with shRNACMAS A549 cells, which are unable to produce sialilated\nglycans. We strongly believe that overall, our data confirms that sialic acid is not\nrequired to utilize MHCII as a receptor.”\nAs noted above, could the authors please provide quantitative values such as\nMFI for the neuraminidase treatment.\nR: As per your request, we have added a new Supplementary Figure 3d and\nSupplementary Figure 7b comparing the MFI values of each sample.\n4. “Replace deacetylated with “desialylated” - you can deacetylate Neu5Ac (the\nacetyl group is on C5), but that’s not what neuraminidase does, you’d need an\nesterase to deacetylate.\nR: Thank you for pointing this out. We have corrected this mistake throughout the\nmanuscript.”\nPlease double check this is correct throughout (for example supplemental figure\n4 and figure 1 legend).\nR: Thank you for pointing this out. After thorough review of the text, we have\nfixed this issue.\n5. Although the authors mention in response to Reviewer 1 that\nA/California/04/2009(H1N1) is a suitable negative control for the specificity of\ntheir assays, could the authors please explain why a positive control was not\nincluded in their experiments? If this is not feasible, I would encourage the\nauthors to elaborate on that in their response.\nR: The positive controls previously suggested were either H17 or H18 strains,\nknown to only use MHCII as an entry receptor. The purpose of a positive control\nis to verify that an assay can detect a known effect. In our case, the effect of\ninterest, MHCII-dependent replication, is directly demonstrated by the viruses\ntested in this study. Both sOH/04 and hVIC/11 replicate in MHCII+, sialic acid–\ndeficient A549 cells, and this phenotype is lost if MHCII is not present, thereby\nfunctioning as internal positive controls within the same experimental system.\nThe central experimental question is binary: Can the viruses infect and replicate\nwhen sialic acid is absent in presence of MHCII? Because sOH/04 and hVIC/11\ndo so robustly, the assay set up is inherently validated without requiring an\nexternal, unrelated virus. We further believe that the inclusion of a well-defined\nnegative control (A/California/04/2009) is more informative in this context. Its\ninability to infect sialic acid–deficient cells demonstrates that the observed effect\nis specific, not due to residual sialic acid or other nonspecific factors, and\nconfirms the specificity of MHCII-mediated entry in our system.\n6. Line 100 in the revised manuscript refers to PAMS that were collected from naïve\npigs in a previous study. Do the authors really mean they used cells that had\nbeen previously collected perhaps frozen or do they mean collected as in a\nprevious study? Please clarify.\nR: We collected samples in previous study that were frozen as previously\ndescribed and retrieved for this study. Line 102-104 now reads: “To further\ninvestigate the role of MHCII during FLUAV, we performed infections using\nalveolar macrophages collected and stored from naïve pigs in a previous study”\n7. Line 179: the words used ‘observed around 70%’ are confusing please rephrase\nmore clearly- what is around 70%- were 70% of the cells expressing SLA-DR?\nR: We have modified the text accordingly, and now reads: “SLA-DR and HLA-DR\nexpression was consistently observed on 70% of the cells on average”\n8. Line 246 the authors use the term ‘on line,’ would ‘in line with’ be better?\nR: Thank you for your comment. We have modified the text as per your\nsuggestion.\n9. Line 259 the authors use the word ‘remanent’, ‘remaining’ or ‘residual’ would be\nclearer.\nR: We have modified the text accordingly, and now reads: “This suggests that\nthe remaining SA was not enough to initiate an infection”\n10. Throughout the manuscript the authors refer to both replication and entry\ninterchangeably. It would be useful for the overall clarity of the manuscript if the\nauthors could please define these terms and be more precise with their use. For\nexample, clearly define how each assay was read out in the text (such as the\nTCID50 equivalent standard curve in Figure 3) and explain how this relates to a\nproductive infection. In Figures 2 and 3, with HA-positive or vRNA readouts, these\nassays seem to provide evidence for entry and transcription, but not necessarily\nproductive replication and infectious progeny, as in Figure 1.\nR: Thank you for pointing this out. We respectfully disagree. In Figure 2, we use\nthe term infection because this experiment captures a single snapshot of infected\ncells at a defined timepoint, and as you noted, it does not assess active\nreplication. In contrast, Figure 3 measures replication, as we performed multi-\ncycle growth kinetics across the different A549 cell groups. Regarding the\nreporting of viral titers, we have found that expressing titers as TCID50\nequivalents provides a more representative measure when using RT-qPCR. This\napproach quantifies viral loads relative to a standard curve generated from a\npreviously titrated TCID50 stock, allowing Ct values to be interpreted in\nbiologically meaningful units. We have modified the text and now We have\napplied this method in multiple prior publications 3,14, and it is the same approach\nused to quantify samples in Figure 1. However, following your advice, we have\ntightened the language and referred to replication only when describing growth\nkinetic curves and have incorporate the following sentence in the M&M section\nlines 620-622: “Time-points were collected at 0, 12, 24, 48, and 72 hours post\ninfection, and viral titers were determined by RT-qPCR using a standard curve\ngenerated from 10-fold serial dilutions of a virus stock with a known TCID50 titer”\nReferences\nhuman and avian H2N2 influenza A viruses. Nat Microbiol (2024).\nhttps://doi.org:10.1038/s41564-024-01771-1\nAdaptive Mutations on the HA Gene. J Virol 96, e0148022 (2022).\nhttps://doi.org:10.1128/jvi.01480-22\nvirus increases affinity for the lower respiratory tract and alveolar macrophages in\npigs. PLoS Pathog. 20, e1012026 (2024).\nhttps://doi.org:10.1371/journal.ppat.1012026\nType D Strain CN3718 Differ in Their Properties and Sensitivities to Inhibitors. Appl\nEnviron Microb 80, 1701-1709 (2014). https://doi.org:10.1128/Aem.03440-13\nenzymatic characterization of the sialidases of Clostridium perfringens. J Biol\nChem 300, 107750 (2024). https://doi.org:10.1016/j.jbc.2024.107750\ncharacterising lectin and antibody specificities in a microarray format. J Biol\nChem, 110915 (2025). https://doi.org:10.1016/j.jbc.2025.110915\nrequired for binding to anti-Neu5Gc antibody. Sci Rep 7, 20029 (2016).\nhttps://doi.org:10.1038/srep20029\nInfluenza A virus in swine alters glycan binding and sialic acid avidity. bioRxiv,\n2025.2012.2010.693614 (2025). https://doi.org:10.64898/2025.12.10.693614\nreceptors for influenza viruses. Proc. Natl. Acad. Sci. U. S. A. 111, E2241-2250\n(2014). https://doi.org:10.1073/pnas.1323162111\nbalancing protection and pathogenesis. J Gen Virol 98, 2401-2412 (2017).\nhttps://doi.org:10.1099/jgv.0.000922\nmacrophages and influenza virus. Eur Respir Rev 33 (2024).\nhttps://doi.org:10.1183/16000617.0132-2024\nmonocyte-to-macrophage transition. Glycobiology 30, 679-694 (2020).\nhttps://doi.org:10.1093/glycob/cwaa020\ncorrelation with influenza virus infection. PLoS Pathog 9, e1003223 (2013).\nhttps://doi.org:10.1371/journal.ppat.1003223\nthe neuraminidase low-affinity calcium-binding pocket. Commun Biol 7, 1230\n(2024). https://doi.org:10.1038/s42003-024-06928-6"}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have addressed my major concern. Actually, because the swine epithelium is sialic acid deficient, according to credible glycomics analysis of swine airway epithelial cells, I think it remains an open question about whether sialic acids or or MHC serve as the primary receptors in swine. That said however, the added section of the discussion nicely addresses this question.", "reviewer_3": "the authors have adequately addressed all my concerns"}, "author_response": ""}]

Decision letter