Human monoclonal antibody F61 nasal spray effectively protected high-risk populations from SARS-CoV-2 variants during the COVID-19 pandemic from late 2022 to early 2023 in China.

Following the national dynamic zero-COVID strategy adjustment, the utilization of broad-spectrum nasal neutralizing antibodies may offer alternative approach to controlling the outbreak of Omicron variants between late 2022 and early 2023 in China. This study involved an investigator-initiated trial (IIT) to assess the pharmacokinetic, safety and efficacy of the F61 nasal spray. A total of 2,008 participants were randomly assigned to receive F61 nasal spray (24 mg/0.8 mL/dose) or normal saline (0.8 mL/dose) and 1,336 completed the follow-up in the IIT. Minimal absorption of F61 antibody into the bloodstream was detected in individuals receiving F61 nasal spray for seven consecutive days. No treatment-emergent adverse reactions of grade 3 severity or higher were reported. In the one-dose cohort, the 7-day cumulative SARS-CoV-2 infection rate was 79.0% in the F61 group and 82.6% in the placebo group, whereas, in the multiple-dose (once daily for 7 consecutive days) cohort, the rates were 6.55% in the F61 group and 23.83% in the placebo group. The laboratory-confirmed efficacy of F61 was 3.78% (-3.74%-10.75%) in the one-dose cohort and 72.19% (57.33%-81.87%) in the multiple-dose cohort. In the real-world study, 60,225 volunteers in four different regions were administered the F61 nasal spray based on subject's wishes, over 90% efficacy rate was observed against different Omicron variants. The F61 nasal spray, with its favorable safety profile, could be a promising prophylactic monoclonal antibody against SARS-CoV-2 VOCs.

Vaccination with Span, an antigen guided by SARS-CoV-2 S protein evolution, protects against challenge with viral variants in mice.

SARS-CoV-2 continues to accumulate mutations to evade immunity, leading to breakthrough infections after vaccination. How researchers can anticipate the evolutionary trajectory of the virus in advance in the design of next-generation vaccines requires investigation. Here, we performed a comprehensive study of 11,650,487 SARS-CoV-2 sequences, which revealed that the SARS-CoV-2 spike (S) protein evolved not randomly but into directional paths of either high infectivity plus low immune resistance or low infectivity plus high immune resistance. The viral infectivity and immune resistance of variants are generally incompatible, except for limited variants such as Beta and Kappa. The Omicron variant has the highest immune resistance but showed high infectivity in only one of the tested cell lines. To provide cross-clade immunity against variants that undergo diverse evolutionary pathways, we designed a new pan-vaccine antigen (Span). Span was designed by analyzing the homology of 2675 SARS-CoV-2 S protein sequences from the NCBI database before the Delta variant emerged. The refined Span protein harbors high-frequency residues at given positions that reflect cross-clade generality in sequence evolution. Compared with a prototype wild-type (Swt) vaccine, which, when administered to mice, induced serum with decreased neutralization activity against emerging variants, Span vaccination of mice elicited broad immunity to a wide range of variants, including those that emerged after our design. Moreover, vaccinating mice with a heterologous Span booster conferred complete protection against lethal infection with the Omicron variant. Our results highlight the importance and feasibility of a universal vaccine to fight against SARS-CoV-2 antigenic drift.

Structural basis of a two-antibody cocktail exhibiting highly potent and broadly neutralizing activities against SARS-CoV-2 variants including diverse Omicron sublineages.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs), especially the latest Omicron, have exhibited severe antibody evasion. Broadly neutralizing antibodies with high potency against Omicron are urgently needed for understanding the working mechanisms and developing therapeutic agents. In this study, we characterized the previously reported F61, which was isolated from convalescent patients infected with prototype SARS-CoV-2, as a broadly neutralizing antibody against all VOCs including Omicron BA.1, BA.1.1, BA.2, BA.3 and BA.4 sublineages by utilizing antigen binding and cell infection assays. We also identified and characterized another broadly neutralizing antibody D2 with epitope distinct from that of F61. More importantly, we showed that a combination of F61 with D2 exhibited synergy in neutralization and protecting mice from SARS-CoV-2 Delta and Omicron BA.1 variants. Cryo-Electron Microscopy (Cryo-EM) structures of the spike-F61 and spike-D2 binary complexes revealed the distinct epitopes of F61 and D2 at atomic level and the structural basis for neutralization. Cryo-EM structure of the Omicron-spike-F61-D2 ternary complex provides further structural insights into the synergy between F61 and D2. These results collectively indicated F61 and F61-D2 cocktail as promising therapeutic antibodies for combating SARS-CoV-2 variants including diverse Omicron sublineages.

Screening of potent neutralizing antibodies against SARS-CoV-2 using convalescent patients-derived phage-display libraries.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten public health worldwide, the development of effective interventions is urgently needed. Neutralizing antibodies (nAbs) have great potential for the prevention and treatment of SARS-CoV-2 infection. In this study, ten nAbs were isolated from two phage-display immune libraries constructed from the pooled PBMCs of eight COVID-19 convalescent patients. Eight of them, consisting of heavy chains encoded by the immunoglobulin heavy-chain gene-variable region (IGHV)3-66 or IGHV3-53 genes, recognized the same epitope on the receptor-binding domain (RBD), while the remaining two bound to different epitopes. Among the ten antibodies, 2B11 exhibited the highest affinity and neutralization potency against the original wild-type (WT) SARS-CoV-2 virus (KD = 4.76 nM for the S1 protein, IC50 = 6 ng/mL for pseudoviruses, and IC50 = 1 ng/mL for authentic viruses), and potent neutralizing ability against B.1.1.7 pseudoviruses. Furthermore, 1E10, targeting a distinct epitope on RBD, exhibited different neutralization efficiency against WT SARS-CoV-2 and its variants B.1.1.7, B.1.351, and P.1. The crystal structure of the 2B11-RBD complexes revealed that the epitope of 2B11 highly overlaps with the ACE2-binding site. The in vivo experiment of 2B11 using AdV5-hACE2-transduced mice showed encouraging therapeutic and prophylactic efficacy against SARS-CoV-2. Taken together, our results suggest that the highly potent SARS-CoV-2-neutralizing antibody, 2B11, could be used against the WT SARS-CoV-2 and B.1.1.7 variant, or in combination with a different epitope-targeted neutralizing antibody, such as 1E10, against SARS-CoV-2 variants.

MLN4924 suppresses lipopolysaccharide-induced proinflammatory cytokine production in neutrophils in a dose-dependent manner.

Neddylation is a ubiquitination-like pathway. It has been reported that neddylation inhibition with the pharmacological agent MLN4924 potently uppresses lipopolysaccharide (LPS)-induced proinflammatory cytokine production, including tumor necrosis factor (TNF)-α and interleukin (IL)-6, by preventing the degradation of phosphorylated inhibitor of κB (p-IκB) in macrophages. However, whether neddylation serves a similar role in neutrophils remains unknown. In the present study MLN4924 treatment led to the accumulation of P-IκBα in neutrophils as well as the decreased production of TNF-α, IL-6 and IL-1ß in response to LPS, in a dose-dependent manner. The viability of neutrophils was only marginally affected in the same conditions, without statistical significance. Furthermore, the nuclear factor (NF)-κB inhibitor JSH-23 mimicked the effects of MLN4924 in neutrophils, and the inhibitory effects of MLN4924 on LPS-induced proinflammatory cytokine production diminished in the presence of JSH-23. Thus, the results of the present study suggest that neddylation inhibition suppresses neutrophil function by suppressing the NF-κB signaling pathway.

All-trans retinoic acid induces autophagic degradation of ubiquitin-like modifier activating enzyme 3 in acute promyelocytic leukemia cells.

All-trans retinoic acid (ATRA) has demonstrated notable success in the treatment of acute promyelocytic leukemia (APL) by inducing granulocytic differentiation. The underlying mechanisms of ATRA therapeutic effects have not been entirely clarified. Here, we reported that the regulation of neddylation, a ubiquitination-like post-translational modification, was involved in the treatment of ATRA on APL. Treating APL cells with ATRA led to the degradation of UBA3, a subunit of neddylation E1. Lysosome-autophagy pathway but not proteasome pathway was responsible for the degradation of UBA3. Neddylation suppression in APL cells was capable of inducing apoptosis, differentiation and proliferation inhibition, suggesting a pivotal role of neddylation in APL cells. ATRA treatment also led to UBA3 degradation in primary APL cells. Taken together, our findings indicated that neddylation was important to maintain the malignant features of APL cells, and suppression of neddylation was involved in the effects of ATRA on APL cells.

Impaired Autophagy and Defective T Cell Homeostasis in Mice with T Cell-Specific Deletion of Receptor for Activated C Kinase 1.

Autophagy plays a central role in maintaining T cell homeostasis. Our previous study has shown that hepatocyte-specific deficiency of receptor for activated C kinase 1 (RACK1) leads to lipid accumulation in the liver, accompanied by impaired autophagy, but its in vivo role in T cells remains unclear. Here, we report that mice with T cell-specific deletion of RACK1 exhibit normal intrathymic development of conventional T cells and regulatory T (Treg) cells but reduced numbers of peripheral CD4+ and CD8+ T cells. Such defects are cell intrinsic with impaired mitochondrial clearance, increased sensitivity to cell death, and decreased proliferation that could be explained by impaired autophagy. Furthermore, RACK1 is essential for invariant natural T cell development. In vivo, T cell-specific loss of RACK1 dampens concanavalin A-induced acute liver injury. Our data suggest that RACK1 is a key regulator of T cell homeostasis.

RACK1 Promotes Autophagy by Enhancing the Atg14L-Beclin 1-Vps34-Vps15 Complex Formation upon Phosphorylation by AMPK.

Autophagy is essential for maintaining tissue homeostasis. Although adaptors have been demonstrated to facilitate the assembly of the Atg14L-Beclin 1-Vps34-Vps15 complex, which functions in autophagosome formation, it remains unknown whether the autophagy machinery actively recruits such adaptors. WD40-repeat proteins are a large, highly conserved family of adaptors implicated in various cellular activities. However, the role of WD40-repeat-only proteins, such as RACK1, in postnatal mammalian physiology remains unknown. Here, we report that hepatocyte-specific RACK1 deficiency leads to lipid accumulation in the liver, accompanied by impaired Atg14L-linked Vps34 activity and autophagy. Further exploration indicates that RACK1 participates in the formation of autophagosome biogenesis complex upon its phosphorylation by AMPK at Thr50. Thr50 phosphorylation of RACK1 enhances its direct binding to Vps15, Atg14L, and Beclin 1, thereby promoting the assembly of the autophagy-initiation complex. These observations provide insight into autophagy induction and establish a pivotal role for RACK1 in postnatal mammalian physiology.