Safety and neutralization antibody levels of inactivated SARS-CoV-2 vaccine in adult patients with Myasthenia Gravis: a prospective observational cohort study.

BACKGROUND AND OBJECTIVES: Myasthenia gravis (MG) is an autoimmune disease affecting the neuromuscular junction. No cohort study has investigated the efficacy of inactivated vaccines in patients with MG. MATERIALS AND METHODS: This prospective observational cohort study included healthy controls (HCs) and patients with MG with or without immunosuppressive treatment. Vaccination occurred between May and December 2021. Patients with MG were subjected to a clinical scale assessment for disease severity. The neutralization antibodies (Nabs) levels were measured in all participants using the pseudovirus neutralization assay. RESULTS: Twenty-one patients (Female/Male:10/11); age median [interquartile range (IQR)]: 43 [30, 56]) were included in this study. Two patients (2/21) were lost during follow-up after enrollment. No sustained vaccine-related adverse effects occurred in any visit of patients with MG. No exacerbation of MG was observed. Acetylcholine receptor antibody (AChR-Ab) levels showed no statistically significant changes between the first and second visit (median [IQR]: 2.22 [0.99, 2.63] nmol/L vs. 1.54 [1.07, 2.40] nmol/L, p = 0.424). However, levels of AChR-Ab decreased at the third visit (median [IQR]: 2.22 [0.96, 2.70] nmol/L vs. 1.69 [0.70, 1.85] nmol/L, p = 0.011). No statistically significant difference in Nabs levels was found between HCs and patients with MG (median [IQR]: 102.89 [33.13, 293.86] vs. 79.29 [37.50, 141.93], p = 0.147). DISCUSSION: The safety of the SARS-CoV-2 inactivated vaccine was reconfirmed in this study. No significant difference in Nabs level was found between patients with MG and HCs. Nabs levels correlated with AChR-Ab levels before vaccination and ΔAChR-Ab levels.

GSTZ1-1 Deficiency Activates NRF2/IGF1R Axis in HCC via Accumulation of Oncometabolite Succinylacetone.

The IGF1R signaling is important in the malignant progression of cancer. However, overexpression of IGF1R has not been properly assessed in HCC. Here, we revealed that GSTZ1-1, the enzyme in phenylalanine/tyrosine catabolism, is downregulated in HCC, and its expression was negatively correlated with IGF1R. Mechanistically, GSTZ1-1 deficiency led to succinylacetone accumulation, alkylation modification of KEAP1, and NRF2 activation, thus promoting IGF1R transcription by recruiting SP1 to its promoter. Moreover, inhibition of IGF1R or NRF2 significantly inhibited tumor-promoting effects of GSTZ1 knockout in vivo. These findings establish succinylacetone as an oncometabolite, and GSTZ1-1 as an important tumor suppressor by inhibiting NRF2/IGF1R axis in HCC. Targeting NRF2 or IGF1R may be a promising treatment approach for this subset HCC.

Mutation Y453F in the spike protein of SARS-CoV-2 enhances interaction with the mink ACE2 receptor for host adaption.

COVID-19 patients transmitted SARS-CoV-2 to minks in the Netherlands in April 2020. Subsequently, the mink-associated virus (miSARS-CoV-2) spilled back over into humans. Genetic sequences of the miSARS-CoV-2 identified a new genetic variant known as "Cluster 5" that contained mutations in the spike protein. However, the functional properties of these "Cluster 5" mutations have not been well established. In this study, we found that the Y453F mutation located in the RBD domain of miSARS-CoV-2 is an adaptive mutation that enhances binding to mink ACE2 and other orthologs of Mustela species without compromising, and even enhancing, its ability to utilize human ACE2 as a receptor for entry. Structural analysis suggested that despite the similarity in the overall binding mode of SARS-CoV-2 RBD to human and mink ACE2, Y34 of mink ACE2 was better suited to interact with a Phe rather than a Tyr at position 453 of the viral RBD due to less steric clash and tighter hydrophobic-driven interaction. Additionally, the Y453F spike exhibited resistance to convalescent serum, posing a risk for vaccine development. Thus, our study suggests that since the initial transmission from humans, SARS-CoV-2 evolved to adapt to the mink host, leading to widespread circulation among minks while still retaining its ability to efficiently utilize human ACE2 for entry, thus allowing for transmission of the miSARS-CoV-2 back into humans. These findings underscore the importance of active surveillance of SARS-CoV-2 evolution in Mustela species and other susceptible hosts in order to prevent future outbreaks.

A spacer design strategy for CRISPR-Cas12f1 with single-nucleotide polymorphism mutation resolution capability and its application in the mutations diagnosis of pathogens.

Infectious diseases remain a major global issue in public health. It is important to develop rapid, sensitive, and accurate diagnostic methods to detect pathogens and their mutations. Cas12f1 is an exceptionally compact RNA-guided nuclease and have the potential to fulfill the clinical needs. Based on the interaction between crRNA-SSDNA binary sequence and Cas12f1, here, we addressed the essential features that determine the recognition ability of CRISPR-Cas12f1 single-nucleotide polymorphism (SNP), such as the length of spacer region and the base pairing region that determines the trans-cleavage of ssDNA. A fine-tuning spacer design strategy is also proposed to enhance the SNP recognition capability of CRISPR-Cas12f1. The optimized spacer confers the Cas12f1 system a strong SNP identification capability for viral or bacterial drug-resistance mutations, with a specificity ratio ranging from 19.63 to 110.20 and an admirable sensitivity up to 100  copy/µL. Together, the spacer screening and CRISPR-Cas12f1 based SNP identification method, is sensitive and versatile, and will have a wide application prospect in pathogen DNA mutation diagnosis and other mutation profiling.

Distinct immune responses in the early phase to natural SARS-CoV-2 infection or vaccination.

Immune responses elicited by viral infection or vaccination play key roles in the viral elimination and the prevention of reinfection, as well as the protection of healthy persons. As one of the most widely used Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, there have been increasing concerns about the necessity of additional doses of inactivated vaccines, due to the waning immune response several months after vaccination. To further optimize inactivated SARS-CoV-2 vaccines, we compared immune responses to SARS-CoV-2 elicited by natural infection and immunization with inactivated vaccines in the early phase. We observed the lower antibody levels against SARS-CoV-2 spike (S) and nucleocapsid (N) proteins in the early phase of postvaccination with a slow increase, compared to the acute phase of SARS-CoV-2 natural infection. Specifically, IgA antibodies have the most significant differences. Moreover, we further analyzed cytokine expression between these two groups. A wide variety of cytokines presented high expression in the infected individuals, while a few cytokines were elicited by inactivated vaccines. The differences in antibody responses and cytokine levels between natural SARS-CoV-2 infection and vaccination with the inactivated vaccines may provide implications for the optimization of inactivated SARS-CoV-2 vaccines and the additional application of serological tests.

Genome-wide association study of SARS-CoV-2 infection in Chinese population.

Coronavirus disease 2019 (COVID-19) is a global public health concern. The purpose of this study was to investigate the association between genetic variants and SARS-CoV-2 infection and the COVID-19 severity in Chinese population. A total of 256 individuals including 87 symptomatic patients (tested positive for SARS-CoV-2), 84 asymptomatic cases, and 85 close contacts of confirmed patients (tested negative for SARS-CoV-2) were recruited from February 2020 to May 2020. We carried out the whole exome genome sequencing between the individuals and conducted a genetic association study for SARS-CoV-2 infection and the COVID-19 severity. In total, we analyzed more than 100,000 single-nucleotide polymorphisms. The genome-wide association study suggested potential correlation between genetic variability in POLR2A, ANKRD27, MAN1A2, and ERAP1 genes and SARS-CoV-2 infection susceptibility. The most significant gene locus associated with SARS-CoV-2 infection was located in POLR2A (p = 5.71 × 10-6). Furthermore, genetic variants in PCNX2, CD200R1L, ZMAT3, PLCL2, NEIL3, and LINC00700 genes (p < 1 × 10-5) were closely associated with the COVID-19 severity in Chinese population. Our study confirmed that new genetic variant loci had significant association with SARS-CoV-2 infection and the COVID-19 severity in Chinese population, which provided new clues for the studies on the susceptibility of SARS-CoV-2 infection and the COVID-19 severity. These findings may give a better understanding on the molecular pathogenesis of COVID-19 and genetic basis of heterogeneous susceptibility, with potential impact on new therapeutic options.

CRISPR/Cas9 delivery by NIR-responsive biomimetic nanoparticles for targeted HBV therapy.

BACKGROUND: Currently, there are no curative drugs for hepatitis B virus (HBV). Complete elimination of HBV covalently closed circular DNA (cccDNA) is key to the complete cure of hepatitis B virus infection. The CRISPR/Cas9 system can directly destroy HBV cccDNA. However, a CRISPR/Cas9 delivery system with low immunogenicity and high efficiency has not yet been established. Moreover, effective implementation of precise remote spatiotemporal operations in CRISPR/Cas9 is a major limitation. RESULTS: In this work, we designed NIR-responsive biomimetic nanoparticles (UCNPs-Cas9@CM), which could effectively deliver Cas9 RNP to achieve effective genome editing for HBV therapy. HBsAg, HBeAg, HBV pgRNA and HBV DNA along with cccDNA in HBV-infected cells were found to be inhibited. These findings were confirmed in HBV-Tg mice, which did not exhibit significant cytotoxicity and minimal off-target DNA damage. CONCLUSIONS: The UCNPs-based biomimetic nanoplatforms achieved the inhibition of HBV replication via CRISPR therapy and it is a potential system for efficient treatment of human HBV diseases.

Longitudinal Dynamics of the Neutralizing Antibody Response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a global pandemic with no licensed vaccine or specific antiviral agents for therapy. Little is known about the longitudinal dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific neutralizing antibodies (NAbs) in patients with COVID-19. METHODS: Blood samples (n = 173) were collected from 30 patients with COVID-19 over a 3-month period after symptom onset and analyzed for SARS-CoV-2-specific NAbs using the lentiviral pseudotype assay, coincident with the levels of IgG and proinflammatory cytokines. RESULTS: SARS-CoV-2-specific NAb titers were low for the first 7-10 days after symptom onset and increased after 2-3 weeks. The median peak time for NAbs was 33 days (interquartile range [IQR], 24-59 days) after symptom onset. NAb titers in 93.3% (28/30) of the patients declined gradually over the 3-month study period, with a median decrease of 34.8% (IQR, 19.6-42.4%). NAb titers increased over time in parallel with the rise in immunoglobulin G (IgG) antibody levels, correlating well at week 3 (r = 0.41, P < .05). The NAb titers also demonstrated a significant positive correlation with levels of plasma proinflammatory cytokines, including stem cell factor (SCF), TNF-related apoptosis-inducing ligand (TRAIL), and macrophage colony-stimulating factor (M-CSF). CONCLUSIONS: These data provide useful information regarding dynamic changes in NAbs in patients with COVID-19 during the acute and convalescent phases.

Dicoumarol, an NQO1 inhibitor, blocks cccDNA transcription by promoting degradation of HBx.

BACKGROUND & AIMS: Current antiviral therapies help keep HBV under control, but they are not curative, as they are unable to eliminate the intracellular viral replication intermediate termed covalently closed circular DNA (cccDNA). Therefore, there remains an urgent need to develop strategies to cure CHB. Functional silencing of cccDNA is a crucial curative strategy that may be achieved by targeting the viral protein HBx. METHODS: We screened 2,000 small-molecule compounds for their ability to inhibit HiBiT-tagged HBx (HiBiT-HBx) expression by using a HiBiT lytic detection system. The antiviral activity of a candidate compound and underlying mechanism of its effect on cccDNA transcription were evaluated in HBV-infected cells and a humanised liver mouse model. RESULTS: Dicoumarol, an inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), significantly reduced HBx expression. Moreover, dicoumarol showed potent antiviral activity against HBV RNAs, HBV DNA, HBsAg and HBc protein in HBV-infected cells and a humanised liver mouse model. Mechanistic studies demonstrated that endogenous NQO1 binds to and protects HBx protein from 20S proteasome-mediated degradation. NQO1 knockdown or dicoumarol treatment significantly reduced the recruitment of HBx to cccDNA and inhibited the transcriptional activity of cccDNA, which was associated with the establishment of a repressive chromatin state. The absence of HBx markedly blocked the antiviral effect induced by NQO1 knockdown or dicoumarol treatment in HBV-infected cells. CONCLUSIONS: Herein, we report on a novel small molecule that targets HBx to combat chronic HBV infection; we also reveal that NQO1 has a role in HBV replication through the regulation of HBx protein stability. LAY SUMMARY: Current antiviral therapies for hepatitis B are not curative because of their inability to eliminate covalently closed circular DNA (cccDNA), which persists in the nuclei of infected cells. HBV X (HBx) protein has an important role in regulating cccDNA transcription. Thus, targeting HBx to silence cccDNA transcription could be an important curative strategy. We identified that the small molecule dicoumarol could block cccDNA transcription by promoting HBx degradation; this is a promising therapeutic strategy for the treatment of chronic hepatitis B.

SIRT7 restricts HBV transcription and replication through catalyzing desuccinylation of histone H3 associated with cccDNA minichromosome.

Chronic hepatitis B virus (HBV) infection is a significant public health burden worldwide. HBV covalently closed circular DNA (cccDNA) organized as a minichromosome in nucleus is responsible for viral persistence and is the key obstacle for a cure of chronic hepatitis B (CHB). Recent studies suggest cccDNA transcription is epigenetically regulated by histone modifications, especially histone acetylation and methylation. In the present study, we identified transcriptionally active histone succinylation (H3K122succ) as a new histone modification on cccDNA minichromosome by using cccDNA ChIP-Seq approach. Silent mating type information regulation 2 homolog 7 (SIRT7), as an NAD+-dependent histone desuccinylase, could bind to cccDNA through interaction with HBV core protein where it catalyzed histone 3 lysine 122 (H3K122) desuccinylation. Moreover, SIRT7 acts cooperatively with histone methyltransferase, suppressor of variegation 3-9 homolog 1 (SUV39H1) and SET domain containing 2 (SETD2) to induce silencing of HBV transcription through modulation of chromatin structure. Our data improved the understanding of histone modifications of the cccDNA minichromosome, thus transcriptional silencing of cccDNA may represent a novel antiviral strategy for the prevention or treatment of HBV infection.