Sicpin, a multifunctional immunomodulatory salivary protein from black flies significantly reduced T and B cell proliferation and directly binds to soluble CD4 receptor

Sicpin, the first immunomodulatory salivary protein described in black flies significantly reduced T and B cell proliferation and directly binds to soluble CD4 receptor Hematophagy is key to blood feeding arthropods reproductive success and an important link in pathogen transmission cycles. Salivary gland homogenates from blackflies have been shown to contain immunomodulatory activity on murine splenocytes. However, the molecule(s) responsible for this salivary activity remains elusive thus far. Here, we report the first immunosuppressive protein from blackfly salivary glands. Sicpin (Simulium cell proliferation inhibitor) was produced in E. coli and purified using size exclusion and ion exchange chromatography. Purified Sicpin was LPS‐decontaminated and its sequence verified by N‐terminal sequencing and LC‐MS/MS analysis. Sicpin inhibited cell proliferation in a dose‐response manner independently of the mitogen utilized (ConA, LPS, CD3/CD28 and Pokeweed). LPS or ConA stimulated cells had a significant lower proliferation rates (P<0.001) in the presence of Sicpin (IC50=0.5μM) with 10uM completely abrogating cell proliferation. Flow cytometry analysis showed that Sicpin inhibited proliferation of CD19+ B‐cells and CD4+/CD8+ T‐cells. Sicpin did not induce apoptosis or necrosis in mitogen‐induced proliferative responses by murine splenocytes as determined by flow cytometry. Sicpin also inhibits antigen‐specific cell proliferation without inducing apoptosis in resting or mitogen‐induced splenocytes. We demonstrate that the production IFN‐α, IL4, IL5, IL6 and IL10 by splenocytes stimulated by ConA or LPS was dose‐dependently reduced by Sicpin. Reduction of cytokines in presence of Sicpin could lead to a retardation of B and T cell activity. Carrageenan‐induced paw‐edema model showed that the intensity of edema significantly decreases when Sicpin was administered at 5 and 15 μg/animal. The molecular mechanism of Sicpin on cell proliferation inhibition is currently under investigation; however, initial binding experiments using SPR analysis showed a direct binding to soluble CD4 receptor with a calculated KD of 17.77 nM. Direct binding of Sicpin to CD4 could inhibit the subsequent TCR ligation‐induced T cell signaling at the earliest steps including tyrosine phosphorylation of the receptors, downstream effector proteins, and lipid raft reorganization. The immune suppressive and anti‐inflammatory properties of Sicpin should be explored as a strategy to modulate immune responses in infection and tumor proliferation as well as its involvement in parasite transmission Support or Funding Information This work was supported by the Intramural Research Program of the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.