The diversity of molecules recognized by the TLR2 receptor may be explained by its capacity to form heterodimers with TLR1, TLR6 or TLR10 and to benefit from the help of additional cofactors including CD14 and CD36 [41]. encouraging non-spike SARS-CoV-2 antigen candidate for inclusion in the development of next-generation prophylactic vaccines against COVID-19 contamination and disease. 0.05, ** for 0.01 and *** for 0.001, while ns means non-significant. 3. Results 3.1. SARS-CoV-2 Envelope (E) Protein Interacts Directly and Physically with TLR2 In order to analyze the capacity of the SARS-CoV-2 envelope (E) protein to interact with TLR2 at a molecular level, a solid-phase assay was used to test the binding of various amounts of E protein (1C1000 ng/mL) to a constant amount of precoated human recombinant TLR2 (1 g/mL). The results show that this E protein bound to TLR2 in a dose-dependent manner (Physique 1A). In contrast, no significant binding to TLR2 was observed when the experiment was performed with GST instead of the E protein (Physique 1A). It was further observed that this E protein, PF-06471553 but not GST used as a control, was also able to bind to human main monocytes (analysis by circulation cytometry, Physique 1B,C) and to human main macrophages (analysis by microscopy, Physique 1D). Open in a separate window Physique 1 Binding of SARS-CoV-2 protein to human TLR2. (A) Soluble recombinant human TLR2 (100 L at 1 g/mL) was coated in 96-well plates. After saturation, numerous amounts of the E-GST protein (1 PF-06471553 ng/mLC1000 ng/mL) were added for 2 h at 37 C. TLR2 E-GST complexes were revealed by a solution of anti-GST sera follow by anti-anti-GST conjugated to HRP. (B) Main human monocytes were incubated with 0.1 to 10 g/mL of GST or GST-E SARS-CoV-2 protein. Cells were stained with anti-GST (1/1000). Data were acquired using a FACScalibur. One representative experiment is shown. (C) Quantification of the SARS-CoV-2 E protein or GST control binding to human monocytes from 3 different experiments acquired using a FACScalibur. (D) Main human macrophages were incubated with 10 g/mL of GST or GST-E SARS-CoV-2 protein. Cells were stained with anti-GST (1/500). Images were acquired using an EVOS M700 microscope. (E) HEK-TLR2 or HEK-null (106) cells were preincubated for 1 h with increasing amounts of inactivated SARS-CoV-2 (0.5C50 g). The staining of SARS-CoV-2 complexes was performed with rabbit anti-spike antibodies used at 1/500 and donkey anti-rabbit IgG complexed with Alexa 488 used at 20 g/mL. (F) HEK-TLR2 or HEK-null cells previously plated in 24-well plates were incubated for 1 h with numerous amounts of inactivated SARS-CoV-2 at RT. After 3 washes, binding of SARS-CoV-2 to HEK cells was evaluated by microscopy, and bound viral particles were detected by anti-spike antibodies. (G) HEK-TLR2 or HEK-null cells previously plated in 24-well plates were incubated for 1 h PF-06471553 with numerous amounts of inactivated SARS-CoV-2 (0.1C100 g) at RT. After washing and cell lysis, cell-associated viral particles were evaluated after SDS-PAGE and Western blotting using anti-spike antibodies. Statistical significance comparing different groups is usually denoted with * PF-06471553 for 0.05, ** for 0.01 and *** for 0.001, while ns means non-significant. In addition, to demonstrate that this native, virus-associated E membrane protein was able to bind to TLR2, the binding of SARS-CoV-2 viral Rabbit Polyclonal to MX2 particles to HEK-TLR2 cell lines was evaluated using three complementary methods. In the first approach, the binding of SARS-CoV-2 to TLR2 was evaluated by circulation cytometry. The results shown in Physique 1E indicate that this SARS-CoV-2 viral particles bound to HEK-TLR2 in a doseCresponse manner, while no significant binding was observed with HEK-null cells. In the second approach, the binding of SARS-CoV-2 PF-06471553 to TLR2 was evaluated by microscopy. Staining with anti-spike antibodies exhibited a clear binding of SARS-CoV-2 viral particles to HEK-TLR2 but not to HEK-null cell lines (Physique 1F). In the third approach, SARS-CoV-2 viral particles (0.1C100 g/mL) were incubated with HEK-TLR2 or HEK-null cells, and the bound viral particles were evaluated, after cell lysis, by SDS-PAGE and Western blotting analysis. In these conditions, a specific band with strong staining, having an electrophoretic mobility compatible with the expected molecular mass (180C200 kDa) of the spike protein, was detected.
