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Thanks for the immense and very nice analysis on our preliminary work.

Regarding your negative comments:
1. Validation of lectins-ELISA has been done in the last 10 years and unfortunately, the most elegant approach the field has, it is to compare the results with positive controls (actually very rare in the field, but we did it).
2. Statistical analysis on solid-phase immunoassays of this type are not rational to make. Yes, we calculated the p values and made a statistical analysis (all perfectly solid), but we think these analysis is out of the context for these assays.
3. Yes, we had several positive and negative controls in our experiments (see supporting Figures and comments)
4. For us, pre-prints, in the context of COVID-19, are data to share with the community. Of course we are now working on different in vitro, neutralization assays to confirm our hypothesis.

Main findings
In addition to its primary target, the ACE2 receptor on host cells, the SARS-CoV-2 spike protein and its glycosylated residues may interact with other carbohydrate-binding receptors on innate immune cells. The results of those interactions have not been well described; activation of myeloid cells via these pathways may contribute to hyper-inflammation and thrombogenesis. In order to describe potential interactions between the glycosylated spike protein and conventional carbohydrate-binding proteins on myeloid cells, Chiodo et al. used solid-phase immunoassays to determine the binding specificity of the spike protein to C-type lectins and Siglecs found on immune cells.

The authors surveyed spike protein binding to dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), Langerin, macrophage galactose binding lectin (MGL), the mannose receptor (MR), dectin-1 (used as a negative control), macrophage inducible calcium-dependent lectin receptor (MBL), and the sialic acid-binding immunoglobulin-type lectins (Siglecs-3, 5, 7, 9, 10). The SARS-CoV-2 spike protein showed strong binding to DC-SIGN and MGL, but not to Langerin, MR, and MBL. Of note, DC-SIGN bears an immune-receptor tyrosine-based activation motif (ITAM), suggesting that it is capable of direct signaling. The spike protein also demonstrated selective binding to Siglec-3, 9, and 10, which bear immune-receptor tyrosine-based inhibition motif (ITIM) and have direct immuno-suppressive effects.

The authors extended this method and concept to assess potential interactions between SARS-CoV-2 spike protein and structural components of pathogens that have been cited to contribute to infections secondary to COVID-19. Additional assays using capsular polysaccharides (CPS) of Streptococcus pneumoniae and lipopolysaccharides (LPS) from Pseudomonas aeruginosa, Salmonella typhimurium, and Shigella flexneri revealed that the spike protein can strongly recognize CPS from S. pneumoniae serotypes Sp19F and Sp23F and LPS from P. aeruginosa but not that of the two enteric pathogens.

In summary, these findings point to ACE2-independent recognition of carbohydrate-binding elements on myeloid cells by SARS-CoV-2 spike protein. While the downstream effects of these interactions remain unclear, this study demonstrates that SARS-CoV-2 viral particles have the potential to directly interact with myeloid cells, without using the ACE2 receptor or intracellular TLRs.

Limitations
All experiments described in this report were in vitro experiments that utilized solid-phase immunoassays. No cellular responses were assessed. Moreover, it is unclear if these interactions are different, if the S protein were derived from infected cells in vivo. Therefore, it is difficult to conclude whether recognition of the SARS-CoV-2 spike protein and its carbohydrate residues by lectins or Siglecs typically found on macrophages and dendritic cells results in an immune response that significantly contributes to COVID-19 immuno-pathology. Therefore, the conclusions concerning the immuno-modulatory impact that these ACE2-independent, carbohydrate-based interactions may have are speculative.

Significance
It is known that carbohydrate-binding proteins on myeloid cells facilitate the internalization of pathogens for lysosomal degradation. So, the experiments described here may provide some context for how monocytes and their myeloid derivatives, including monocyte-derived DCs and macrophages, could harbor SARS-CoV-2 particles or viral proteins, despite the lack of evidence of ACE2 receptor expression in these cells. Albeit, it has been hypothesized that ACE2 receptor expression can be induced by interferon in mononuclear phagocytes.

In addition to demonstrating that the spike protein is capable of recognizing certain C-type lectins and Siglecs found on monocyte-derived DCs and macrophages, the authors interrogated the ability for the spike protein to interact with polysaccharide components derived from S. pneumoniae, which has been documented as the most common cause of secondary pneumonia in COVID-19 patients. Additional mechanistic studies in cell culture and in in vivo models are required to verify whether these interactions, in fact, contribute to the immuno-pathology of COVID-19 and viral dissemination.

This review was undertaken by Matthew D. Park as part of a project by students, postdocs and faculty at the Immunology Institute of the Icahn school of medicine, Mount Sinai.