AlphaFold2 assists in providing novel mechanistic insights into the interactions among the LUBAC subunits.

The linear ubiquitin chain assembly complex (LUBAC) is the only known E3 ligase complex in which the ubiquitin-like (UBL) domains of SHARPIN and HOIL-1L interact with HOIP to determine the structural stability of LUBAC. The interactions between subunits within LUBAC have been a topic of extensive research. However, the impact of the LTM motif on the interaction between the UBL domains of SHARPIN and HOIL-1L with HOIP remains unclear. Here, we discover that the absence of the LTM motif in the AlphaFold2-predicted LUBAC structure alters the HOIP-UBA structure. We employ GeoPPI to calculate the changes in binding free energy (ΔG) caused by single-point mutations between subunits, simulating their protein-protein interactions. The results reveal that the presence of the LTM motif decreases the interaction between the UBL domains of SHARPIN and HOIL-1L with HOIP, leading to a decrease in the structural stability of LUBAC. Furthermore, using the AlphaFold2-predicted results, we find that HOIP (629‒695) and HOIP-UBA bind to both sides of HOIL-1L-UBL, respectively. The experiments of Gromacs molecular dynamics simulations, SPR and ITC demonstrate that the elongated domain formed by HOIP (629‒695) and HOIP-UBA, hereafter referred to as the HOIP (466‒695) structure, interacts with HOIL-1L-UBL to form a structurally stable complex. These findings illustrate the collaborative interaction between HOIP-UBA and HOIP (629‒695) with HOIL-1L-UBL, which influences the structural stability of LUBAC.

Pre-Clinical Development of a Potent Neutralizing Antibody MW3321 With Extensive SARS-CoV-2 Variants Coverage.

Since the outbreak of the coronavirus disease 2019 (COVID-19) pandemic, several variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged and have consistently replaced the previous dominant variant. Therapeutics against variants of SARS-CoV-2 are urgently needed. Ideal SARS-CoV-2 therapeutic antibodies would have high potency in viral neutralization against several emerging variants. Neutralization antibodies targeting SARS-CoV-2 could provide immediate protection after SARS-CoV-2 infection, especially for the most vulnerable populations. In this work, we comprehensively characterize the breadth and efficacy of SARS-CoV-2 RBD-targeting fully human monoclonal antibody (mAb) MW3321. MW3321 retains full neutralization activity to all tested 12 variants that have arisen in the human population, which are assigned as VOC (Variants of Concern) and VOI (Variants of Interest) due to their impacts on public health. Escape mutation experiments using replicating SARS-CoV-2 pseudovirus show that escape mutants were not generated until passage 6 for MW3321, which is much more resistant to escape mutation compared with another clinical staged SARS-CoV-2 neutralizing mAb MW3311. MW3321 could effectively reduce viral burden in hACE2-transgenic mice challenged with either wild-type or Delta SARS-CoV-2 strains through viral neutralization and Fc-mediated effector functions. Moreover, MW3321 exhibits a typical hIgG1 pharmacokinetic and safety profile in cynomolgus monkeys. These data support the development of MW3321 as a monotherapy or cocktail against SARS-CoV-2-related diseases.

Antibody-dependent enhancement (ADE) of SARS-CoV-2 pseudoviral infection requires FcγRIIB and virus-antibody complex with bivalent interaction.

Understanding the underlying molecular mechanisms behind ADE of SARS-CoV-2 is critical for development of safe and effective therapies. Here, we report that two neutralizing mAbs, MW01 and MW05, could enhance the infection of SARS-CoV-2 pseudovirus on FcγRIIB-expressing B cells. X-ray crystal structure determination and S trimer-binding modeling showed that MW01 and MW05 could bind to RBDs in S trimer with both "up" and "down" states. While, the neutralizing mAb MW07, which has no ADE activity only binds to RBD in S trimer with "up" state. Monovalent MW01 and MW05 completely diminished the ADE activity compared with their bivalent counterparts. Moreover, both macropinocytosis and endocytosis are confirmed involving in ADE of SARS-CoV-2 pseudoviral infection. Blocking endosome transportation and lysosome acidification could inhibit the ADE activity mediated by MW05. Together, our results identified a novel ADE mechanism of SARS-CoV-2 pseudovirus in vitro, FcγRIIB-mediated uptake of SARS-CoV-2/mAb complex with bivalent interaction.

Characterization of neutralizing antibody with prophylactic and therapeutic efficacy against SARS-CoV-2 in rhesus monkeys.

Efficacious interventions are urgently needed for the treatment of COVID-19. Here, we report a monoclonal antibody (mAb), MW05, with SARS-CoV-2 neutralizing activity by disrupting the interaction of receptor binding domain (RBD) with angiotensin-converting enzyme 2 (ACE2) receptor. Crosslinking of Fc with FcγRIIB mediates antibody-dependent enhancement (ADE) activity by MW05. This activity is eliminated by introducing the LALA mutation to the Fc region (MW05/LALA). Potent prophylactic and therapeutic effects against SARS-CoV-2 are observed in rhesus monkeys. A single dose of MW05/LALA blocks infection of SARS-CoV-2 in prophylactic treatment and clears SARS-CoV-2 in three days in a therapeutic treatment setting. These results pave the way for the development of MW05/LALA as an antiviral strategy for COVID-19.

T0001, a variant of TNFR2-Fc fusion protein, exhibits improved Fc effector functions through increased binding to membrane-bound TNFα.

T0001 is a recombinant human TNFR-Fc fusion protein mutant; it exhibits higher affinity to TNFα than etanercept and is now being tested in a Phase 1 study in China (ClinicalTrials.gov Identifier: NCT02481180). T0001 can inhibit the binding of soluble TNFα (sTNFα) or membrane-bound TNFα (mTNFα) to TNF receptors. When bound to mTNFα, the Fc-bearing TNFα antagonists have the potential to induce Fc-mediated effects, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) as well as outside-to-inside signals (apoptosis mainly). Recent studies have shown that ADCC may also play an important role in Crohn's disease (CD) and ulcerative colitis (UC). In this study, T0001 presented a higher binding activity on mTNFα than etanercept and similar binding activity with adalimumab and infliximab. Upon the addition of sTNFα, adalimumab and infliximab showed significantly increased binding to FcγRIIIa and C1q than T0001 and etanercept. T0001 exhibited significantly higher ADCC and CDC activity than etanercept, and the potency and the reporter response of T0001 were very close to adalimumab and infliximab in ADCC reporter gene assays. And the similar potency of T0001 was also corroborated by PMBC-based ADCC assay. T0001, but not etanercept could induce apoptosis, while adalimumab and infliximab were more effective. These results suggest that T0001 may not only exert improved efficacy in treating rheumatoid arthritis (RA) because of its high affinity to sTNFα but also has a therapeutic potential in CD and UC due to its increased binding to mTNFα with resultant Fc-associated functions (ADCC, in particular) and improved apoptosis.

Distribution and hepatocellular carcinoma-related viral properties of hepatitis B virus genotypes in Mainland China: a community-based study.

INTRODUCTION: Hepatitis B virus (HBV) genotypes, replication status, and mutations have been associated with the risk of hepatocellular carcinoma (HCC). Our aim was to study the distribution and HCC-related viral properties of HBV genotypes/subgenotypes in Mainland China. METHODS: A multistage cluster probability sampling method was applied to select 81,775 participants between 1 and 59 years at 160 national disease surveillance points. We examined hepatitis B surface antigen, HBV genotypes and subgenotypes, hepatitis B e antigen, viral load, and mutations in the PreS and core promoter regions of HBV genome. RESULTS: HBV subgenotypes B2 (27.3%), C1 (10.7%), and C2 (58.0%) were predominant. Genotype D (D1, 80.8%) was frequent in the Uygur. We identified a new subgenotype, C9, mainly in Tibetans. Compositions of subgenotypes B2 and C1 and genotype mixture increased from the North to Central South, which was consistently associated with the increasing prevalence of hepatitis B surface antigen. Hepatitis B e antigen positivity and viral loads were higher in the young with genotype B and declined more rapidly with increasing age than those with genotype C. In contrast to G1896A, PreS deletion, T31C, T1753V, and A1762T/G1764A were more frequent in subgenotype C2 than in subgenotype B2. A1762T/G1764A, T1753V, C1653T, and G1896A, except PreS deletion, consecutively increased with increasing age. CONCLUSION: HBV subgenotypes B2, C1, and C2 are endemic in Mainland China. HBV genotype C exhibits less replication activity in the young and harbors higher frequencies of the HCC-associated mutations than genotype B. IMPACT: These basic data could help evaluate the association of HBV variations with HCC.

Associations between hepatitis B virus mutations and the risk of hepatocellular carcinoma: a meta-analysis.

BACKGROUND: The association between hepatitis B virus (HBV) mutations and hepatocarcinogenesis remains controversial because of conflicting data in the literature. We conducted a meta-analysis of case-control and cohort studies to examine HBV PreS, enhancer II (EnhII), basal core promoter (BCP), and precore mutations in relation to the risk of hepatocellular carcinoma (HCC). METHODS: We searched databases for studies of these associations that were published in English or Chinese up to August 31, 2008. HBV mutation-specific odds ratios and relative risks were pooled by use of a random-effects model and stratified by potential confounders. All statistical tests were two-sided. RESULTS: Of the 43 studies included in this meta-analysis, 40 used a case-control design. The 43 studies evaluated a total of 11 582 HBV-infected participants, of whom 2801 had HCC. Statistically significant summary odds ratios of HCC were obtained for any PreS mutation (3.77, 95% confidence interval [CI] = 2.57 to 5.52), C1653T in EnhII (2.76, 95% CI = 2.09 to 3.64), T1753V (2.35, 95% CI = 1.63 to 3.40), and A1762T/G1764A in BCP (3.79, 95% CI = 2.71 to 5.29). PreS mutations were more strongly associated with an increased risk of HCC in subjects who were infected with HBV genotype C than in those who were infected with HBV genotype B, whereas the opposite was true for A1762T/G1764A. C1653T, T1753V, and A1762T/G1764A were more strongly associated with an increased risk of HCC in hepatitis B e antigen (HBeAg)-positive subjects than in HBeAg-negative subjects. PreS mutations, C1653T, T1753V, and A1762T/G1764A accumulated during the progression of chronic HBV infection from the asymptomatic carrier state to HCC (P(trend) < .001 for each mutation). PreS mutations, C1653T, C1653T + T1753V, and A1762T/G1764A-based combinations of mutations had specificities greater than 80% for the prediction of HCC. The precore mutations G1896A and C1858T were not associated with the risk of HCC, regardless of HBeAg status and HBV genotype. CONCLUSIONS: HBV PreS mutations, C1653T, T1753V, and A1762T/G1764A are associated with an increased risk of HCC. These mutations alone and in combination may be predictive for hepatocarcinogenesis.

An outbreak of dengue virus serotype 1 infection in Cixi, Ningbo, People's Republic of China, 2004, associated with a traveler from Thailand and high density of Aedes albopictus.

Autochthonous dengue infections have not been reported in Ningbo, People's Republic of China since 1929. In August-October 2004, an outbreak of dengue fever was confirmed in Xiaolin, Cixi, Ningbo. Of 83 cases reported, 68 were laboratory confirmed. Fifty-three percent (34 of 64) of the cases had IgM antibodies to dengue virus. Dengue virus serotype-1 was isolated from two cases. The outbreak was linked to a traveler who returned from Thailand. Phylogenetic analysis showed that the Ningbo isolate was closely associated to strains from Thailand. Prevalence of dengue-specific IgG in asymptomatic residents was significantly higher in the epidemic-stricken area than in a control area. High density of Aedes albopictus, which resulted from waterlogging caused by Typhoon Rananim and lifestyle of local residents, was responsible for rapid spread of the virus. Eradication of mosquito infestation might interrupt transmission. This outbreak underscores the importance of maintaining surveillance and control of potential vectors for the control of emerging infectious diseases.