Immunoglobulin M (IgM) delays disease in K18-hACE2 mice infected with SARS-CoV-2
Preeti Chhabra1, Barbara Mann2, Mingyang Ma1, Sanford Feldman3, Kenneth Brayman1.
1Surgery, University of Virginia, Charlottesville, VA, United States; 2Infectious Diseases, University of Virginia, Charlottesville, VA, United States; 3Center for Comparative Medicine, University of Virginia, Charlottesville, VA, United States
Introduction: IgM reverses new onset autoimmune type 1 diabetes and promotes graft survival by mitigating inflammation. It reprograms the immune system by inducing regulatory T cells, promoting the expression of microRNAs associated with the upregulation of regulatory immune cells, and repopulating dysregulated gut microbiome with beneficial bacteria. IgM also prevents HIV-1 viral entry into cells and protects mice from influenza virus infection and strep-pneumococcal infection. Therefore, the goal was to determine if IgM can delay or prevent disease in SARS-CoV-2 infected K18-hACE2 mice.
Method: 1) Vero E6 cells were used to test the effect of IgM in reducing the number of plaque-forming units (PFU).There were 4 groups: a) 25PFU WA-1 SARS-CoV-2 was combined with 20, 5 or 0.8μg IgM in growth medium, and added to Vero E6 cells b) IgM was added to Vero E6 cells and incubated. The media was aspirated, and the cells were inoculated with 25PFU WA-1; c) Virus control - as above, but with no IgM; d) No virus or IgM. Following incubation with virus for 48 hours, virus replication was stopped and plates stained with Giemsa violet, dried, and photographed. 2) A COVID -19 Spike-ACE2 binding assay kit was used to determine if IgM (2ug, 4.5ug, 20ug, 45ug IgM) inhibited the interaction between the Spike-receptor binding domain (S-RBD) and Angiotensin I ConvertingEnzyme2 (ACE2) receptor. 3) K18-hACE2 mice were divided into 3 groups based on treatment regimen; Group 1: with IgM, No virus; 2: with Saline, with virus; 3: with IgM, with virus. 35ug IgM was injected intraperitoneal in a single dose, 2 days prior to infection. Mice were innoculated intranasally with 1250 pfu of HK SARS-CoV-2.
Results: 1) Exposure of 25PFU SARS-CoV-2 to IgM (at all concentrations) prior to incubation with Vero E6 cells, inhibited its replication in Vero E6 cells. When Vero E6 cells were incubated with IgM prior to infection, no plaques were seen in wells with 20ug and 5ug IgM but were observed in wells with 0.8ug IgM. Plaques were also observed in the Virus alone group, but none were seen in the ‘No IgM-No virus’ group. 2) 45ug IgM/100uls inhibited the binding of S-RBD toACE2 by ~94-100%, 20ug IgM/100uls inhibited it by ~80%, and 2 or 4.5ug/100ul by ~70-75%. Control without IgM did not inhibit the S-RBD─ACE-2 binding. 3) Pretreatment with a single low dose IgM injection delayed weight loss and mortality.
Conclusion: IgM inhibits the replication of SARS-CoV-2 in Vero cells in vitro. It also inhibits the interaction between S-RBD that is present on the viral surface and the ACE2 receptor, by binding to S-RBD. A single low dose of IgM given prechallenge delayed disease in infected mice. The discovery that IgM interferes with the formation of the S-RBD─ACE2 complex, and that a single low dose can delay disease, indicates its translational potential as a vaccine/therapeutic to prevent or treat COVID-19.
We would like to acknowledge the "Focus to Cure Diabetes Foundation" which has enabled us to do this research.
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