Vaccination with SARS-CoV-2 spike protein lacking glycan shields elicits enhanced protective responses in animal models.

A major challenge to end the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is to develop a broadly protective vaccine that elicits long-term immunity. As the key immunogen, the viral surface spike (S) protein is frequently mutated, and conserved epitopes are shielded by glycans. Here, we revealed that S protein glycosylation has site-differential effects on viral infectivity. We found that S protein generated by lung epithelial cells has glycoforms associated with increased infectivity. Compared to the fully glycosylated S protein, immunization of S protein with N-glycans trimmed to the mono-GlcNAc-decorated state (SMG) elicited stronger immune responses and better protection for human angiotensin-converting enzyme 2 (hACE2) transgenic mice against variants of concern (VOCs). In addition, a broadly neutralizing monoclonal antibody was identified from SMG-immunized mice that could neutralize wild-type SARS-CoV-2 and VOCs with subpicomolar potency. Together, these results demonstrate that removal of glycan shields to better expose the conserved sequences has the potential to be an effective and simple approach for developing a broadly protective SARS-CoV-2 vaccine.

Capsid proteins of foot-and-mouth disease virus interact with TLR2 and CD14 to induce cytokine production.

The mechanism of recognition of the foot-and-mouth disease virus (FMDV) by host innate immune cells is not well-understood. In this study, we first found that binary ethylenimine inactivated-FMDV (BEI-FMDV) with structurally intact capsid activated TLR2, but not other TLRs, and this specific activation was blocked by anti-TLR2 Abs or knockout of TLR2. BEI-FMDV activated NF-κB to induce cytokines, notably interferon-ß and IL-6, in a TLR2 and MyD88-dependent manner. Coexpression of TLR6 and CD14 showed additive effects on BEI-FMDV/TLR2-mediated activation of NF-κB. Further studies demonstrated that recombinant capsid proteins rVP1 and rVP3 of FMDV but not rVP0 bound directly with CD14 and TLR2. The rVP1- and rVP3-mediated activation of TLR2 and NF-κB were enhanced by the coexpression of TLR1 or TLR6. Immunoprecipitation of either rVP1 or rVP3 with mouse macrophage cell extracts revealed that rVP1 or rVP3 associated with TLR2, CD14 and TLR6 suggesting that rVP1 and rVP3 interact with CD14, TLR2/TLR1, and TLR2/TLR6 heterodimer. Additional study confirmed that rVP1 and rVP3 interacted with the swine TLR2 signaling pathway to induce IL-6 in swine macrophages. Our results identify VP1 and VP3 of FMDV as novel TLR agonists whose recognition by CD14, TLR2/TLR1, and TLR2/TLR6 of host innate immune cells is critical for the induction of cytokine production.

HGK/MAP4K4 deficiency induces TRAF2 stabilization and Th17 differentiation leading to insulin resistance.

Proinflammatory cytokines play important roles in insulin resistance. Here we report that mice with a T-cell-specific conditional knockout of HGK (T-HGK cKO) develop systemic inflammation and insulin resistance. This condition is ameliorated by either IL-6 or IL-17 neutralization. HGK directly phosphorylates TRAF2, leading to its lysosomal degradation and subsequent inhibition of IL-6 production. IL-6-overproducing HGK-deficient T cells accumulate in adipose tissue and further differentiate into IL-6/IL-17 double-positive cells. Moreover, CCL20 neutralization or CCR6 deficiency reduces the Th17 population or insulin resistance in T-HGK cKO mice. In addition, leptin receptor deficiency in T cells inhibits Th17 differentiation and improves the insulin sensitivity in T-HGK cKO mice, which suggests that leptin cooperates with IL-6 to promote Th17 differentiation. Thus, HGK deficiency induces TRAF2/IL-6 upregulation, leading to IL-6/leptin-induced Th17 differentiation in adipose tissue and subsequent insulin resistance. These findings provide insight into the reciprocal regulation between the immune system and the metabolism.

DUSP4 deficiency enhances CD25 expression and CD4+ T-cell proliferation without impeding T-cell development.

The differentiation and activation of T cells are critically modulated by MAP kinases, which are in turn feed-back regulated by dual-specificity phosphatases (DUSPs) to determine the duration and magnitude of MAP kinase activation. DUSP4 (also known as MKP2) is a MAP kinase-induced DUSP member that is dynamically expressed during thymocyte differentiation. We generated DUSP4-deficient mice to study the function of DUSP4 in T-cell development and activation. Our results show that thymocyte differentiation and activation-induced MAP kinase phosphorylation were comparable between DUSP4-deficient and WT mice. Interestingly, activated DUSP4(-/-) CD4(+) T cells were hyperproliferative while DUSP4(-/-) CD8(+) T cells proliferated normally. Further mechanistic studies suggested that the hyperproliferation of DUSP4(-/-) CD4(+) T cells resulted from enhanced CD25 expression and IL-2 signaling through increased STAT5 phosphorylation. Immunization of DUSP4(-/-) mice recapitulated the T-cell hyperproliferation phenotype in antigen recall responses, while the profile of Th1/Th2-polarized antibody production was not altered. Overall, these results suggest that other DUSPs may compensate for DUSP4 deficiency in T-cell development, MAP kinase regulation, and Th1/Th2-mediated antibody responses. More importantly, our data indicate that DUSP4 suppresses CD4(+) T-cell proliferation through novel regulations in STAT5 phosphorylation and IL-2 signaling.