Characterization of a Novel ACE2-Based Therapeutic with Enhanced Rather than Reduced Activity against SARS-CoV-2 Variants.

The human angiotensin-converting enzyme 2 acts as the host cell receptor for SARS-CoV-2 and the other members of the Coronaviridae family SARS-CoV-1 and HCoV-NL63. Here, we report the biophysical properties of the SARS-CoV-2 spike variants D614G, B.1.1.7, B.1.351, and P.1 with affinities to the ACE2 receptor and infectivity capacity, revealing weaknesses in the developed neutralizing antibody approaches. Furthermore, we report a preclinical characterization package for a soluble receptor decoy engineered to be catalytically inactive and immunologically inert, with broad neutralization capacity, that represents an attractive therapeutic alternative in light of the mutational landscape of COVID-19. This construct efficiently neutralized four SARS-CoV-2 variants of concern. The decoy also displays antibody-like biophysical properties and manufacturability, strengthening its suitability as a first-line treatment option in prophylaxis or therapeutic regimens for COVID-19 and related viral infections. IMPORTANCE Mutational drift of SARS-CoV-2 risks rendering both therapeutics and vaccines less effective. Receptor decoy strategies utilizing soluble human ACE2 may overcome the risk of viral mutational escape since mutations disrupting viral interaction with the ACE2 decoy will by necessity decrease virulence, thereby preventing meaningful escape. The solution described here of a soluble ACE2 receptor decoy is significant for the following reasons: while previous ACE2-based therapeutics have been described, ours has novel features, including (i) mutations within ACE2 to remove catalytical activity and systemic interference with the renin/angiotensin system, (ii) abrogated FcγR engagement, reduced risk of antibody-dependent enhancement of infection, and reduced risk of hyperinflammation, and (iii) streamlined antibody-like purification process and scale-up manufacturability indicating that this receptor decoy could be produced quickly and easily at scale. Finally, we demonstrate that ACE2-based therapeutics confer a broad-spectrum neutralization potency for ACE2-tropic viruses, including SARS-CoV-2 variants of concern in contrast to therapeutic MAb.

Survival of mature T cells in the periphery is intrinsically dependent on GIMAP1 in mice.

An effective immune system depends upon the survival of mature T cells in the periphery. Members of the GIMAP family of GTPases have been proposed to regulate this homeostasis, supported by the paucity of peripheral T cells in rodents deficient for either GIMAP1 or GIMAP5. It is unclear whether this lack of T cells is a consequence of an ontological defect, causing the thymus to generate and export T cells incapable of surviving in the periphery, or whether (alternatively or additionally) mature T cells intrinsically require GIMAP1 for survival. Using the ERT2 Cre+ transgene, we conditionally deleted Gimap1 in C57BL/6 mice and demonstrate that GIMAP1 is intrinsically required for the survival of mature T cells in the periphery. We show that, in contrast to GIMAP5, this requirement is independent of the T-cells' activation status. We investigated the nature of the survival defect in GIMAP1-deficient CD4+ T cells and show that the death occurring after GIMAP1 ablation is accompanied by mitochondrial depolarization and activation of the extrinsic apoptotic pathway. This study shows that GIMAP1 is critical for maintaining the peripheral T-cell pool in mice and offers a potent target for the treatment of T-cell-mediated diseases.

GIMAP1 Is Essential for the Survival of Naive and Activated B Cells In Vivo.

An effective immune system depends upon regulation of lymphocyte function and homeostasis. In recent years, members of the GTPases of the immunity associated protein (GIMAP) family were proposed to regulate T cell homeostasis. In contrast, little is known about their function and mode of action in B cells. We used a combination of transgenic mice and in vivo and in vitro techniques to conditionally and electively ablate GIMAP1 in resting and activated peripheral B cells. Our data suggest that GIMAP1 is absolutely essential for the survival of peripheral B cells, irrespective of their activation state. Together with recent data showing increased expression of GIMAP1 in B cell lymphomas, our work points to the possible potential of GIMAP1 as a target for manipulation in a variety of B cell-mediated diseases.

Gata3 directly regulates early inner ear expression of Fgf10.

The analysis of Fgf10 mouse mutants has demonstrated a critical role for this ligand in neurosensory development of the vertebrate inner ear, and we have been looking to define the direct upstream regulators of Fgf10 in this sensory organ, as part of constructing the programme of early inner ear development. Through the analysis of reporter constructs in transgenic mouse embryos and neonatal mice, in this report we define a minimal 1400 bp enhancer from the 5' flanking region of Fgf10. This enhancer drives reporter transgene expression in a manner that recapitulates endogenous expression of Fgf10, from its initial onset in the invaginating otic placode and onwards throughout gestation, controlling Fgf10 expression in all developing sensory patches and in the developing VIIIth ganglion. This regulatory region includes three putative Gata3 binding sites that we demonstrate directly interacts with Gata3 protein through the DNA binding domain with differing affinities. Site directed mutagenesis of all three sites and functional testing in transgenic embryos using reporter transgenes reveals an absolute requirement for Gata3 in controlling Fgf10 expression. Transgenic analysis of individual Gata3 binding site mutations illustrates that only one of these binding sites is necessary for reporter expression. Together these data demonstrate that Gata3 directly activates Fgf10 in the early inner ear, and does so through a single binding site.

Aberrant chromatin landscape following loss of the H3.3 chaperone Daxx in haematopoietic precursors leads to Pu.1-mediated neutrophilia and inflammation.

Defective silencing of retrotransposable elements has been linked to inflammageing, cancer and autoimmune diseases. However, the underlying mechanisms are only partially understood. Here we implicate the histone H3.3 chaperone Daxx, a retrotransposable element repressor inactivated in myeloid leukaemia and other neoplasms, in protection from inflammatory disease. Loss of Daxx alters the chromatin landscape, H3.3 distribution and histone marks of haematopoietic progenitors, leading to engagement of a Pu.1-dependent transcriptional programme for myelopoiesis at the expense of B-cell differentiation. This causes neutrophilia and inflammation, predisposing mice to develop an autoinflammatory skin disease. While these molecular and phenotypic perturbations are in part reverted in animals lacking both Pu.1 and Daxx, haematopoietic progenitors in these mice show unique chromatin and transcriptome alterations, suggesting an interaction between these two pathways. Overall, our findings implicate retrotransposable element silencing in haematopoiesis and suggest a cross-talk between the H3.3 loading machinery and the pioneer transcription factor Pu.1.