Metastable HIV-1 Surface Protein Env Sensitizes Cell Membranes to Transformation and Poration by Dual-Acting Virucidal Entry Inhibitors.

Dual-acting virucidal entry inhibitors (DAVEIs) have previously been shown to cause irreversible inactivation of HIV-1 Env-presenting pseudovirus by lytic membrane transformation. This study examined whether this transformation could be generalized to include membranes of Env-presenting cells. Flow cytometry was used to analyze HEK293T cells transiently transfected with increasing amounts of DNA encoding JRFL Env, loaded with calcein dye, and treated with serial dilutions of microvirin (Q831K/M83R)-DAVEI. Comparing calcein retention against intact Env expression (via Ab 35O22) on individual cells revealed effects proportional to Env expression. "Low-Env" cells experienced transient poration and calcein leakage, while "high-Env" cells were killed. The cell-killing effect was confirmed with an independent mitochondrial activity-based cell viability assay, showing dose-dependent cytotoxicity in response to DAVEI treatment. Transfection with increasing quantities of Env DNA showed further shifts toward "High-Env" expression and cytotoxicity, further reinforcing the Env dependence of the observed effect. Controls with unlinked DAVEI components showed no effect on calcein leakage or cell viability, confirming a requirement for covalently linked DAVEI compounds to achieve Env transformation. These data demonstrate that the metastability of Env is an intrinsic property of the transmembrane protein complex and can be perturbed to cause membrane disruption in both virus and cell contexts.

Restricted HIV-1 Env glycan engagement by lectin-reengineered DAVEI protein chimera is sufficient for lytic inactivation of the virus.

We previously reported a first-generation recombinant DAVEI construct, a dual action virus entry inhibitor composed of cyanovirin-N (CVN) fused to a membrane proximal external region or its derivative peptide Trp3. DAVEI exhibits potent and irreversible inactivation of HIV-1 (human immunodeficiency virus) viruses by dual engagement of gp120 and gp41. However, the promiscuity of CVN to associate with multiple glycosylation sites in gp120 and its multivalency limit current understanding of the molecular arrangement of the DAVEI molecules on trimeric spike. Here, we constructed and investigated the virolytic function of second-generation DAVEI molecules using a simpler lectin, microvirin (MVN). MVN is a monovalent lectin with a single glycan-binding site in gp120, is structurally similar to CVN and exhibits no toxicity or mitogenicity, both of which are liabilities with CVN. We found that, like CVN-DAVEI-L2-3Trp (peptide sequence DKWASLWNW), MVN-DAVEI2-3Trp exploits a similar mechanism of action for inducing HIV-1 lytic inactivation, but by more selective gp120 glycan engagement. By sequence redesign, we significantly increased the potency of MVN-DAVEI2-3Trp protein. Unlike CVN-DAVEI2-3Trp, re-engineered MVN-DAVEI2-3Trp(Q81K/M83R) virolytic activity and its interaction with gp120 were both competed by 2G12 antibody. That the lectin domain in DAVEIs can utilize MVN without loss of virolytic function argues that restricted HIV-1 Env (envelope glycoprotein) glycan engagement is sufficient for virolysis. It also shows that DAVEI lectin multivalent binding with gp120 is not required for virolysis. MVN-DAVEI2-3Trp(Q81K/M83R) provides an improved tool to elucidate productive molecular arrangements of Env-DAVEI enabling virolysis and also opens the way to form DAVEI fusions made up of gp120-binding small molecules linked to Trp3 peptide.

Evaluation of Adult Patients Readmitted for Severe Sepsis/Septic Shock Under the BPCI Advanced Program.

Bundled Payments for Care Improvement-Advanced Program (BPCI-A) is designed to pay a single payment covering services provided during an episode of care. Sepsis is associated with increased readmissions, mortality, and health care costs. The purpose of the study was to evaluate the BPCI program patients with sepsis who were readmitted within 90 days versus not readmitted. This was a retrospective cohort study including 271 (110 readmitted) patients enrolled in the BPCI program with Diagnostic-Related Grouping codes of septicemia or severe sepsis. Skin/soft tissue infection was the most common infection. There was a significant difference between the groups for resource needs at discharge including wound care (25.45% versus 11.18%; P = 0.002) and physical therapy (74.55% versus 57.14%; P = 0.004). Mortality was higher among readmissions, 43.64% versus 26.71% no readmission ( P = 0.004). Identifying risk factors for readmission, providing appropriate resources, and follow-up may contribute to improved patient outcomes for patients with sepsis enrolled in the BPCI program.