African Green Monkey Cell Line (Vero E6) and Baby hamster kidney cells (BHK-21, 12-14-17 MAW, Kerafast, Boston, MA) were cultured in Dulbecco´s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 25 μg/mL gentamycin, and 2 mM L-glutamine.

Blood samples were obtained from healthy human donors (Hospital Universitario 12 de Octubre, Madrid, Spain) subject to informed consent and IRB approval. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation (Ficoll Paque, GE17-5442-02, Sigma-Aldrich). To generate monocyte-derived dendritic cells (MDDCs), CD14+ monocytes were purified using anti-human CD14 antibody-labeled magnetic beads and iron-based LS columns (Miltenyi Biotec) and used directly for further differentiation into dendritic cells (Domínguez-Soto et al., 2011). Differentiation to immature MDDCs was achieved by incubation at 37°C with 5% CO2 for 7 days and subsequent activation with cytokines GM-CSF (1000 U/mL) and IL-4 (500 U/mL) (Miltenyi Biotec) every other day.

rVSV-luc pseudotypes (PSV) were generated following a published protocol (Whitt, 2010). First, BHK-21cells were transfected using Lipofectamine 3000 (Fisher Scientific) to express the different viral envelope glycoproteins: Ebola virus Makona Glycoprotein (EBOV-GP, GenBank KM233102.1) and S protein of MERS-CoV (NC_019843) were synthesized and cloned into pcDNA3.1 by GeneArt AG technology (Life Technologies, Regensburg, Germany); S protein of SARS-CoV-2 (codon-optimized; kindly provided by J. García-Arriaza, CNB-CSIC, Spain) or S protein of SARS-CoV (SinoBiological, Cat: VG40150-G-N) were cloned into a pCMV/hygro vector. After 24 h, transfected cells were inoculated with a replication-deficient rVSV-luc pseudotype (MOI: 3–5) (Kerafast, Boston, MA). After 1 h incubation at 37°C, cells were washed exhaustively with PBS and then DMEM supplemented with 5% heat-inactivated FBS, and 25 μg/mL gentamycin and 2 mM L-glutamine were added. PSV were collected 24 h post-inoculation, clarified from cellular debris by centrifugation, and stored at -80°C. Infectious titers were estimated as tissue culture infectious doses by limiting dilution of rVSV-luc-pseudotypes on Vero E6 cells. Luciferase activity was determined by luciferase assay (Steady-Glo Luciferase Assay System, Promega).

The antibodies used in the experiments were Mouse IgG2a Isotype Control (Invitrogen), MERS Coronavirus Spike Protein neutralizing Antibody MA5-29975 (Invitrogen), Invitrogen CD26 Monoclonal Antibody (BA5b) (Invitrogen), Human DC-SIGN+DC-SIGNR Antibody (R&D Systems), PE anti-human CD209 (DC-SIGN) Antibody (Biolegend), and CD26 Monoclonal Antibody (BA5b) PE (Invitrogen). As a lectin-binding control, mannan from Saccharomyces cerevisiae (Sigma-Aldrich) was used. Mannan is used in experiments to inhibit C-type lectin receptors, such as DC-SIGN due to its ability to block the interaction between these receptors and viral or pathogenic ligands. Being a complex polysaccharide, mannan competes with the binding sites of the receptors, acting as a decoy and preventing the attachment of infectious agents to host cells (Jan et al., 2018).

primary cells MDDCs (5x10⁴) were challenged with MERS-CoV, SARS-CoV, SARS-CoV-2, EBOV-GP pseudotyped recombinant viruses (MOI: 0.5–2), mannan, and different antibodies ( Figure 1 ). After 24 h of incubation, cells were washed twice with PBS and lysed for luciferase assay.

For trans-infection studies, MDDCs (5x10⁴) were challenged with recombinant MERS-CoV, SARS-CoV, SARS-CoV-2, or EBOV-GP pseudotyped viruses (MOI: 0.5–2) and incubated for 2h with rotation ( Figure 2 ). The antibodies were incubated with the MDDC cells 40 minutes prior to virus exposure, except for the neutralizing antibody against MERS, which was incubated with the virus for 40 minutes before the addition of the cells. After the incubation time, cells were then centrifuged at 1200 rpm for 5 minutes and washed six times with PBS supplemented with 0.5% bovine serum albumin (BSA) and 1 mM CaCl2 to ensure the removal of unbound virus from the cell receptor. The inclusion of CaCl2 in the wash buffer helps to maintain the integrity and functionality of DC-SIGN during the experimental procedures. After that, MDDCs were then resuspended in RPMI medium and co-cultivated with adherent Vero E6 cells (1.5x10⁵ cells/well) on a 24-well plate. After 24h, the supernatant was removed and the monolayer of Vero E6 was washed with PBS six times and lysed for luciferase assay. These washes were performed to remove the co-cultivated dendritic cells from the Vero E6 cells and ensure that the infection observed in the Vero E6 cells was due to the trans-infection process and not direct infection that may occur in the MDDC cells. In order to control this variable, a parallel trans-infection experiment with MERS-CoV was also conducted on MDDC cells under the same conditions. After the incubation period, the dendritic cells were not co-cultivated with the Vero E6 target cells following the washes with PBS. The levels of RLU/s in the infection readout after 24 hours indicate the amount of MERS-CoV that had successfully entered through direct infection in the dendritic cells during the two-hour incubation period.

The duration of migration for a dendritic cell from the mucosal layers where MERS-CoV could potentially adhere to a deeper tissue where infection initiation occurs remains uncertain. To enhance our comprehension of this biological phenomenon, a trans-infection assay was conducted, incorporating varying time intervals between attachment and infection. In order to monitor the viral entry of MERS-CoV into MDDCs during these incubation periods, an infection assay was carried out under identical conditions. Trypsin treatment: To discern between viral attachment and entry into the cell, a trypsin treatment experiment (Hanley et al., 2010) was conducted on MDDCs both before and after exposure to the MERS-CoV virus. This was performed to ascertain whether trans-infection was stopped.

MERS-CoV PSV demonstrated a clear capability to infect MDDC in cis, and levels of infection were reduced in the presence of anti-DC/L-SIGN although it was not significant, and it was reduced by mannan ( Figure 1 ). Cis infection of MDDC with SARS-CoV-1 and SARS-CoV-2 PSV displayed a total absence of infection, whereas EBOV PSV exhibited clear infection of MDDC that was inhibited by anti-DC-SIGN as demonstrated before (Alvarez et al., 2002).

The isotype control antibody employed alongside the anti-DC/L-SIGN and anti-DPP4 antibodies confirmed the specificity of the primary antibody binding, ruling out any potential effects from other protein interactions or nonspecific Fc receptor binding. Cis infection by MERS-CoV in the presence of anti-DPP4 antibody exhibited a significant reduction, thereby demonstrating the utilization as a direct cellular receptor by MERS-CoV. Infection with MERS-CoV was completely abolished in the presence of its neutralizing antibody ( Figure 3 ).

The results showed percentages (%) considering cis-infection in the absence of antibodies as 100% of infection.

To evaluate the effectiveness of these antibodies, a cis infection assay was conducted in human 293T cells and African green monkey Vero E6 cells.

The results showed that 293T cells were infected by MERS-CoV and SARS-CoV-2. MERS-CoV and SARS-CoV-2 infections in 293T were not inhibited by isotype control antibody; however, infection with MERS-CoV in the presence of the anti-DPP4 antibody showed a significant reduction, unlike SARS-CoV-2 where the infection persisted. Additionally, MERS-CoV infection has been inhibited in the presence of the neutralizing antibody ( Supplementary Figure 1 ).

On the other hand, cis-infection showed that Vero E6 cells were infected by MERS-CoV and SARS-CoV-2 and this infection was not inhibited by isotype control antibody. Infection with MERS-CoV in the presence of the anti-DPP4 antibody did not show a significant reduction due to the anti-DPP4 antibody being a human antibody. Additionally, MERS-CoV infection has been inhibited in the presence of the neutralizing antibody ( Supplementary Figure 2 ).

The trans-infection assay in MDDC with MERS-CoV PSV led to high levels of infection and anti-DC-SIGN exhibited a significant reduction in infectivity showing that DC-SIGN promoted an efficient trans-infection by MERS-CoV from MDDC to Vero E6 cells ( Figure 4 ). Similar DC-SIGN-specific trans-infection was also shown for SARS-CoV, SARS-CoV-2, and EBOV PSV.

An isotype control antibody was used for both anti-DC/L-SIGN and anti-DPP4 antibodies to confirm that the binding of the primary antibody is specific and not the result of other protein interactions or non-specific binding of the Fc receptor. The trans-infection mediated by MERS-CoV was entirely suppressed when its neutralizing antibody was present, and the control of inhibition of infection mediated by lectin receptors mannan reduced the trans-infection. Finally, trans-infection by MERS-CoV in the presence of the anti-DPP4 antibody did not show a significant reduction.

On a separate note, to control the viral entry of MERS-CoV into MDDCs during these incubation periods, a concurrent trans-infection experiment involving MDDC cells was also carried out using identical conditions. Subsequent to the incubation phase, the dendritic cells were not co-cultured with the Vero E6 target cells after PBS washes. The levels of relative light units per second (RLU/s) in the infection readout after 24 hours demonstrated minimal infection by MERS-CoV, in comparison with the levels of trans-infection in Vero E6 ( Supplementary Figure 3 ).

To further explore the role of DC-SIGN as a trans-receptor for MERS-CoV, an infection assay was conducted under identical conditions at different time points. The assay demonstrated effective trans-infection at 2, 6, and 12 hours, with the highest binding observed at the six-hour mark. The parallel assay in MDDCs revealed that viral entry into the cells increases with the incubation time, albeit remaining modest in comparison to the levels of trans-infection observed in Vero E6 cells ( Supplementary Figure 4 ).

The results of the trypsin treatment experiment followed by PBS washing demonstrated that trans-infection is halted when MDDCs are treated both before exposure to the MERS-CoV virus and after virus attachment. There were no significant differences between pre-incubation and post-incubation trypsin treatment during the trans-infection assay ( Supplementary Figure 5 ).

PE anti-human CD209 (DC-SIGN) Antibody (Biolegend) and CD26 Monoclonal Antibody (BA5b) PE (Invitrogen) were employed to quantify the expression of the DC-SIGN receptor ( Supplementary Figure 6 ) and DPP4 receptor ( Supplementary Figure 7 ) on Monocyte-Derived Dendritic Cells (MDDCs). The expression was analyzed by flow cytometry in a Cytek Aurora with FlowJo V10.7.1 software.