Soluble Expression of a Neo2/15-Conjugated Single Chain Fv against PD-L1 in Escherichia coli

Immunocytokines, antibody-cytokine fusion proteins, have the potential to improve the therapeutic index of cytokines by delivering the cytokine to the site of localized tumor cells using antibodies. In this study, we produced a recombinant anti-programmed death-ligand 1 (PD-L1) scFv, an antibody fragment against PD-L1 combined with a Neo2/15, which is an engineered interleukin with superior function using an E. coli expression system. We expressed the fusion protein in a soluble form and purified it, resulting in high yield and purity. The high PD-L1-binding efficiency of the fusion protein was confirmed via enzyme-linked immunosorbent assay, suggesting the application of this immunocytokine as a cancer-related therapeutic agent.

Recombinant Fusion Protein Vaccine Containing FliC and FliD Protects Mice Against Clostridioides Difficile Infection

Bacterial flagella are involved in infection through their roles in host-cell adhesion, cell invasion, auto-agglutination, colonization, and formation of biofilms, as well as in the regulation and secretion of non-flagellar bacterial proteins involved in the virulence process. In this study, we constructed a fusion protein vaccine (FliCD) containing Clostridiodes difficile flagellar proteins FliC and FliD. Immunization of mice with FliCD induce potent IgG antibody responses against FliCD and protected mice against C. difficile infection and decrease C. difficile spores and toxin levels in the feces after infection. Furthermore, we found anti-FliCD serum protected mice against CDI and decreased C. difficile spores and toxin levels in the feces after C. difficile infection. Finally, we found that anti-FliCD serum inhibited the binding of C. difficile vegetative cells to HCT8 cells. These results imply that FliCD fusion protein may represent an effective vaccine candidate for the prevention from C. difficile infection (CDI).

Nanobody-Functionalized Cellulose for Capturing SARS-CoV-2

The highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 253 million people, claiming ∼ 5.1 million lives to date. Although mandatory quarantines, lockdowns, and vaccinations help curb viral transmission, there is a pressing need for cost-effective systems to mitigate the viral spread. Here, we present a generic strategy for capturing SARS-CoV-2 through functionalized cellulose materials. Specifically, we developed a bifunctional fusion protein consisting of a cellulose-binding domain and a nanobody (Nb) targeting the receptor-binding domain of SARS-CoV-2. The immobilization of the fusion proteins on cellulose substrates enhanced the capture efficiency of Nbs against SARS-CoV-2 pseudoviruses of the wildtype and the D614G variant, the latter of which has been shown to confer higher infectivity. Furthermore, the fusion protein was integrated into a customizable chromatography with highly porous cellulose to capture viruses from complex fluids in a continuous fashion. By capturing and containing viruses through the Nb-functionalized cellulose, our work may find utilities in virus sampling and filtration towards paper-based diagnostics, environmental tracking of viral spread and reducing viral load from infected individuals. IMPORTANCE The ongoing efforts to address the COVID-19 pandemic center around the development of diagnostics, preventative measures, and therapeutic strategies. In comparison to existing work, we have provided a complementary strategy to capture SARS-CoV-2 by functionalized cellulose materials towards paper-based diagnostics as well as virus filtration in perishable samples. Specifically, we developed a bifunctional fusion protein consisting of both a cellulose-binding domain and a nanobody specific for the receptor-binding domain of SARS-CoV-2. As a proof-of-concept, the fusion protein-coated cellulose substrates exhibited enhanced capture efficiency against SARS-CoV-2 pseudovirus of both wildtype and the D614G mutant variants, the latter of which has been shown to confer higher infectivity. Furthermore, the fusion protein was integrated into a customizable chromatography for binding viruses from complex biological fluids in a highly continuous and cost-effective manner. Such antigen-specific capture can potentially immobilize viruses of interest for viral detection and removal, which contrasts with the common size- or affinity-based filtration devices that bind a broad range of bacteria, viruses, fungi, and cytokines present in blood ( https://clinicaltrials.gov/ct2/show/NCT04413955 ). Additionally, since our work focuses on capturing and concentrating viruses from surfaces and fluids as a means to improve detection, it can serve as an “add-on” technology to complement existing viral detection methods, many of which have been largely focusing on improving the intrinsic sensitivities.

A bacterium-like particle vaccine displaying Zika virus prM-E induces systemic immune responses in mice

The emergence of Zika virus (ZIKV) infection, which is unexpectedly associated with congenital defects, has prompted the development of safe and effective vaccines. The gram-positive enhancer matrix-protein anchor (GEM-PA) display system has emerged as a versatile and highly effective platform for delivering target proteins for vaccines. In this article, we developed a bacterium-like particle vaccine ZI-△-PA-GEM based on the GEM-PA system. The fusion protein ZI-△-PA, which contains the prM-E-△ protein of ZIKV (with a stem-transmembrane region deletion) and the protein anchor PA3, was expressed. The fusion protein was successfully displayed on the GEM surface, forming ZI-△-PA-GEM. Moreover, when BALB/c mice were immunized intramuscularly with ZI-△-PA-GEM combined with 201 VG and poly(I:C) adjuvants, durable ZIKV-specific IgG and protective neutralizing antibody responses were induced. Potent B cell/DC activation was also be stimulated early after immunization. Remarkably, splenocyte proliferation, the secretion of multiple cytokines, T/B cell activation and central memory T cell responses were elicited. These data indicate that ZI-△-PA-GEM is a promising bacterium-like particle vaccine candidate for ZIKV.

Effective Menin inhibitor-based combinations against AML with MLL rearrangement or NPM1 mutation (NPM1c)

Treatment with Menin inhibitor (MI) disrupts the interaction between Menin and MLL1 or MLL1-fusion protein (FP), inhibits HOXA9/MEIS1, induces differentiation and loss of survival of AML harboring MLL1 re-arrangement (r) and FP, or expressing mutant (mt)-NPM1. Following MI treatment, although clinical responses are common, the majority of patients with AML with MLL1-r or mt-NPM1 succumb to their disease. Pre-clinical studies presented here demonstrate that genetic knockout or degradation of Menin or treatment with the MI SNDX-50469 reduces MLL1/MLL1-FP targets, associated with MI-induced differentiation and loss of viability. MI treatment also attenuates BCL2 and CDK6 levels. Co-treatment with SNDX-50469 and BCL2 inhibitor (venetoclax), or CDK6 inhibitor (abemaciclib) induces synergistic lethality in cell lines and patient-derived AML cells harboring MLL1-r or mtNPM1. Combined therapy with SNDX-5613 and venetoclax exerts superior in vivo efficacy in a cell line or PD AML cell xenografts harboring MLL1-r or mt-NPM1. Synergy with the MI-based combinations is preserved against MLL1-r AML cells expressing FLT3 mutation, also CRISPR-edited to introduce mtTP53. These findings highlight the promise of clinically testing these MI-based combinations against AML harboring MLL1-r or mtNPM1.

Fusion protein strategies for cryo-EM study of G protein-coupled receptors

Single particle cryogenic-electron microscopy (cryo-EM) is used extensively to determine structures of activated G protein-coupled receptors (GPCRs) in complex with G proteins or arrestins. However, applying it to GPCRs without signaling proteins remains challenging because most receptors lack structural features in their soluble domains to facilitate image alignment. In GPCR crystallography, inserting a fusion protein between transmembrane helices 5 and 6 is a highly successful strategy for crystallization. Although the similar strategy has the potential to broadly facilitate cryo-EM structure determination of GPCRs alone without signaling protein, the critical determinants that make this approach successful are not yet clear. Here, we address this shortcoming by exploring different fusion protein designs, which led to structures of antagonist bound A2A adenosine receptor at 3.4&Aring resolution and unliganded Smoothened at 3.7&Aring resolution. The fusion strategies explored here are likely applicable to cryo-EM interrogation of other GPCRs and small integral membrane proteins.