Sinovac Biotech

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented public health crisis. Because of the novelty of the virus, there are currently no SARS-CoV-2-specific treatments or vaccines available. Therefore, rapid development of effective vaccines against SARS-CoV-2 are urgently needed. Here, we developed a pilot-scale production of PiCoVacc, a purified inactivated SARS-CoV-2 virus vaccine candidate, which induced SARS-CoV-2-specific neutralizing antibodies in mice, rats, and nonhuman primates. These antibodies neutralized 10 representative SARS-CoV-2 strains, suggesting a possible broader neutralizing ability against other strains. Three immunizations using two different doses, 3 or 6 micrograms per dose, provided partial or complete protection in macaques against SARS-CoV-2 challenge, respectively, without observable antibody-dependent enhancement of infection. These data support the clinical development and testing of PiCoVacc for use in humans.

In the past few decades, hundreds of materials have been tried as adjuvant; however, only aluminum-based adjuvants continue to be used widely in the world. Aluminum hydroxide, aluminum phosphate and alum constitute the main forms of aluminum used as adjuvants. Among these, aluminum hydroxide is the most commonly used chemical as adjuvant. In spite of its wide spread use, surprisingly, the mechanism of how aluminum hydroxide-based adjuvants exert their beneficial effects is still not fully understood. Current explanations for the mode of action of aluminum hydroxide-based adjuvants include, among others, the repository effect, pro-phagocytic effect, and activation of the pro-inflammatory NLRP3 pathway. These collectively galvanize innate as well as acquired immune responses and activate the complement system. Factors that have a profound influence on responses evoked by aluminum hydroxide-based adjuvant applications include adsorption rate, strength of the adsorption, size and uniformity of aluminum hydroxide particles, dosage of adjuvant, and the nature of antigens. Although vaccines containing aluminum hydroxide-based adjuvants are beneficial, sometimes they cause adverse reactions. Further, these vaccines cannot be stored frozen. Until recently, aluminum hydroxide-based adjuvants were known to preferentially prime Th2-type immune responses. However, results of more recent studies show that depending on the vaccination route, aluminum hydroxide-based adjuvants can enhance both Th1 as well as Th2 cellular responses. Advances in systems biology have opened up new avenues for studying mechanisms of aluminum hydroxide-based adjuvants. These will assist in scaling new frontiers in aluminum hydroxide-based adjuvant research that include improvement of formulations, use of nanoparticles of aluminum hydroxide and development of composite adjuvants.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a pandemic with no specific antiviral treatments or vaccines. There is an urgent need for exploring the neutralizing antibodies from patients with different clinical characteristics. METHODS: A total of 117 blood samples were collected from 70 COVID-19 inpatients and convalescent patients. Antibodies were determined with a modified cytopathogenic neutralization assay (NA) based on live severe acute respiratory syndrome coronavirus 2 and enzyme-linked immunosorbent assay (ELISA). The dynamics of neutralizing antibody levels at different time points with different clinical characteristics were analyzed. RESULTS: The seropositivity rate reached up to 100.0% within 20 days since onset, and remained 100.0% till days 41-53. The total geometric mean titer was 1:163.7 (95% confidence interval [CI], 128.5-208.6) by NA and 1:12 441.7 (95% CI, 9754.5-15 869.2) by ELISA. The antibody level by NA and ELISA peaked on days 31-40 since onset, and then decreased slightly. In multivariate generalized estimating equation analysis, patients aged 31-45, 46-60, and 61-84 years had a higher neutralizing antibody level than those aged 16-30 years (β = 1.0470, P = .0125; β = 1.0613, P = .0307; β = 1.3713, P = .0020). Patients with a worse clinical classification had a higher neutralizing antibody titer (β = 0.4639, P = .0227). CONCLUSIONS: The neutralizing antibodies were detected even at the early stage of disease, and a significant response was shown in convalescent patients.

Early diagnosis of colon cancer (CC) is clinically important, as it can significantly improve patients' survival rate and quality of life. Although the potential role for small extracellular vesicles (sEVs) in early detection of many diseases has been repeatedly mentioned, systematic screening of plasma sEVs derived early CC specific biomarkers has not yet been reported. In this work, plasma sEVs enriched fractions were derived from 15 early-stage (TisN0M0) CC patients and 10 normal controls (NC). RNA sequencing identified a total number of 95 sEVs enriched fraction derived miRNAs with differential expression between CC and NC, most of which (60/95) was in well accordance with tissue results in the Cancer Genome Atlas (TCGA) dataset. Among those miRNAs, we selected let-7b-3p, miR-139-3p, miR-145-3p, and miR-150-3p for further validation in an independent cohort consisting of 134 participants (58 CC and 76 NC). In the validation cohort, the AUC of 4 individual miRNAs ranged from 0.680 to 0.792. A logistic model combining two miRNAs (i.e. let-7b-3p and miR-145-3p) achieved an AUC of 0.901. Adding the 3rd miRNA into this model can further increase the AUC to 0.927. Side by side comparison revealed that sEVs miRNA profile outperformed cell-free plasma miRNA in the diagnosis of early CC. In conclusion, we suggested that circulating sEVs enriched fractions have a distinct miRNA profile in CC patients, and sEVs derived miRNA could be used as a promising biomarker to detect CC at an early stage.

Abstract Purpose: Autologous chimeric antigen receptor T (CAR-T) cell therapy is an effective treatment for relapsed/refractory acute lymphoblastic leukemia (r/r ALL). However, certain characteristics of autologous CAR-T cells can delay treatment availability. Relapse caused by antigen escape after single-targeted CAR-T therapy is another issue. Therefore, we aim to develop CRISPR-edited universal off-the-shelf CD19/CD22 dual-targeted CAR-T cells as a novel therapy for r/r ALL. Patients and Methods: In this open-label dose-escalation phase I study, universal CD19/CD22-targeting CAR-T cells (CTA101) with a CRISPR/Cas9-disrupted TRAC region and CD52 gene to avoid host immune-mediated rejection were infused in patients with r/r ALL. Safety, efficacy, and CTA101 cellular kinetics were evaluated. Results: CRISPR/Cas9 technology mediated highly efficient, high-fidelity gene editing and production of universal CAR-T cells. No gene editing–associated genotoxicity or chromosomal translocation was observed. Six patients received CTA101 infusions at doses of 1 (3 patients) and 3 (3 patients) × 106 CAR+ T cells/kg body weight. Cytokine release syndrome occurred in all patients. No dose-limiting toxicity, GvHD, neurotoxicity, or genome editing–associated adverse events have occurred to date. The complete remission (CR) rate was 83.3% on day 28 after CTA101 infusion. With a median follow-up of 4.3 months, 3 of the 5 patients who achieved CR or CR with incomplete hematologic recovery (CR/CRi) remained minimal residual disease (MRD) negative. Conclusions: CRISPR/Cas9-engineered universal CD19/CD22 CAR-T cells exhibited a manageable safety profile and prominent antileukemia activity. Universal dual-targeted CAR-T cell therapy may offer an alternative therapy for patients with r/r ALL.

. Vaccination strategies to counter immune imprinting are critically needed. Here we investigated the degree and dynamics of immune imprinting in mouse models and human cohorts, especially focusing on the role of repeated Omicron stimulation. In mice, the efficacy of single Omicron boosting is heavily limited when using variants that are antigenically distinct from WT-such as the XBB variant-and this concerning situation could be mitigated by a second Omicron booster. Similarly, in humans, repeated Omicron infections could alleviate WT vaccination-induced immune imprinting and generate broad neutralization responses in both plasma and nasal mucosa. Notably, deep mutational scanning-based epitope characterization of 781 receptor-binding domain (RBD)-targeting monoclonal antibodies isolated from repeated Omicron infection revealed that double Omicron exposure could induce a large proportion of matured Omicron-specific antibodies that have distinct RBD epitopes to WT-induced antibodies. Consequently, immune imprinting was largely mitigated, and the bias towards non-neutralizing epitopes observed in single Omicron exposures was restored. On the basis of the deep mutational scanning profiles, we identified evolution hotspots of XBB.1.5 RBD and demonstrated that these mutations could further boost the immune-evasion capability of XBB.1.5 while maintaining high ACE2-binding affinity. Our findings suggest that the WT component should be abandoned when updating COVID-19 vaccines, and individuals without prior Omicron exposure should receive two updated vaccine boosters.

. Here we examined whether sera from individuals who received two or three doses of inactivated SARS-CoV-2 vaccine could neutralize authentic Omicron. The seroconversion rates of neutralizing antibodies were 3.3% (2 out of 60) and 95% (57 out of 60) for individuals who had received 2 and 3 doses of vaccine, respectively. For recipients of three vaccine doses, the geometric mean neutralization antibody titre for Omicron was 16.5-fold lower than for the ancestral virus (254). We isolated 323 human monoclonal antibodies derived from memory B cells in triple vaccinees, half of which recognized the receptor-binding domain, and showed that a subset (24 out of 163) potently neutralized all SARS-CoV-2 variants of concern, including Omicron. Therapeutic treatments with representative broadly neutralizing monoclonal antibodies were highly protective against infection of mice with SARS-CoV-2 Beta (B.1.351) and Omicron. Atomic structures of the Omicron spike protein in complex with three classes of antibodies that were active against all five variants of concern defined the binding and neutralizing determinants and revealed a key antibody escape site, G446S, that confers greater resistance to a class of antibodies that bind on the right shoulder of the receptor-binding domain by altering local conformation at the binding interface. Our results rationalize the use of three-dose immunization regimens and suggest that the fundamental epitopes revealed by these broadly ultrapotent antibodies are rational targets for a universal sarbecovirus vaccine.

BACKGROUND: A number of oncoproteins and tumor suppressors are known to be neddylated, but whether the neddylation pathway is entirely activated in human cancer remains unexplored. METHODS: NEDD8-activating enzyme (NAE) (E1) and NEDD8-conjugating enzyme (E2) expression and global-protein neddylation were examined by immunohistochemistry, immunoblotting, and real-time polymerase chain reaction analysis. Cell proliferation, clonogenic survival, migration, and motility in vitro, as well as tumor formation and metastasis in vivo, were determined upon neddylation inhibition by MLN4924, an investigational NEDD8-activating enzyme inhibitor. Survival was analyzed with Kaplan-Meier methods and compared by the log-rank test. All statistical tests were two-sided. RESULTS: The entire neddylation pathway, including NEDD8-activating enzyme E1, NEDD8-conjugating enzyme E2, and global-protein neddylation, is overactivated in both lung adenocarcinoma and squamous-cell carcinoma. Compared with lung adenocarcinoma patients with low expression, those with high expression had worse overall survival (NEDD8-activating enzyme E1 subunit 1 [NAE1]: hazard ratio [HR] = 2.07, 95% confidence interval [CI] = 0.95 to 4.52, P = .07; ubiquitin-conjugating enzyme E2M (UBC12): HR = 13.26, 95% CI = 1.77 to 99.35, P = .01; global protein neddylation: HR = 3.74, 95% CI = 1.65 to 8.47, P = .002). Moreover, inhibition of neddylation by the NAE inhibitor MLN4924 statistically significantly suppressed proliferation, survival, migration, and motility of lung cancer cells in vitro and tumor formation and metastasis in vivo. At the molecular level, MLN4924 inactivated Cullin-RING E3 ligases, led to accumulation of tumor-suppressive Cullin-RING E3 ligase substrates and induced phorbol-12-myristate-13-acetate-induced protein 1 (NOXA)-dependent apoptosis or cellular senescence. CONCLUSIONS: Our study highlights the overactivated neddylation pathway in lung cancer development and as a promising therapeutic target.

Synthetic biology aims to design or assemble existing bioparts or bio-components for useful bioproperties. During the past decades, progresses have been made to build delicate biocircuits, standardized biological building blocks and to develop various genomic/metabolic engineering tools and approaches. Medical and pharmaceutical demands have also pushed the development of synthetic biology, including integration of heterologous pathways into designer cells to efficiently produce medical agents, enhanced yields of natural products in cell growth media to equal or higher than that of the extracts from plants or fungi, constructions of novel genetic circuits for tumor targeting, controllable releases of therapeutic agents in response to specific biomarkers to fight diseases such as diabetes and cancers. Besides, new strategies are developed to treat complex immune diseases, infectious diseases and metabolic disorders that are hard to cure via traditional approaches. In general, synthetic biology brings new capabilities to medical and pharmaceutical researches. This review summarizes the timeline of synthetic biology developments, the past and present of synthetic biology for microbial productions of pharmaceutics, engineered cells equipped with synthetic DNA circuits for diagnosis and therapies, live and auto-assemblied biomaterials for medical treatments, cell-free synthetic biology in medical and pharmaceutical fields, and DNA engineering approaches with potentials for biomedical applications.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sublineages have escaped most receptor-binding domain (RBD)-targeting therapeutic neutralizing antibodies (NAbs), which proves that previous NAb drug screening strategies are deficient against the fast-evolving SARS-CoV-2. Better broad NAb drug candidate selection methods are needed. Here, we describe a rational approach for identifying RBD-targeting broad SARS-CoV-2 NAb cocktails. Based on high-throughput epitope determination, we propose that broad NAb drugs should target non-immunodominant RBD epitopes to avoid herd-immunity-directed escape mutations. Also, their interacting antigen residues should focus on sarbecovirus conserved sites and associate with critical viral functions, making the antibody-escaping mutations less likely to appear. Following these criteria, a featured non-competing antibody cocktail, SA55+SA58, is identified from a large collection of broad sarbecovirus NAbs isolated from SARS-CoV-2-vaccinated SARS convalescents. SA55+SA58 potently neutralizes ACE2-utilizing sarbecoviruses, including circulating Omicron variants, and could serve as broad SARS-CoV-2 prophylactics to offer long-term protection, especially for individuals who are immunocompromised or with high-risk comorbidities.

Although several different flaviviruses may cause encephalitis, Japanese encephalitis virus is the most significant, being responsible for thousands of deaths each year in Asia. The structural and molecular basis of this encephalitis is not fully understood. Here, we report the cryo-electron microscopy structure of mature Japanese encephalitis virus at near-atomic resolution, which reveals an unusual "hole" on the surface, surrounded by five encephalitic-specific motifs implicated in receptor binding. Glu138 of E, which is highly conserved in encephalitic flaviviruses, maps onto one of these motifs and is essential for binding to neuroblastoma cells, with the E138K mutation abrogating the neurovirulence and neuroinvasiveness of Japanese encephalitis virus in mice. We also identify structural elements modulating viral stability, notably Gln264 of E, which, when replaced by His264 strengthens a hydrogen-bonding network, leading to a more stable virus. These studies unveil determinants of neurovirulence and stability in Japanese encephalitis virus, opening up new avenues for therapeutic interventions against neurotropic flaviviruses.Japanese encephalitis virus (JEV) is a Flavivirus responsible for thousands of deaths every year for which there are no specific anti-virals. Here, Wang et al. report the cryo-EM structure of mature JEV at near-atomic resolution and identify structural elements that modulate stability and virulence.

Abstract Background COVID-19 is a pandemic with no specific antiviral treatments or vaccines. The urgent needs for exploring the neutralizing antibodies from patients with different clinical characteristics are emerging. Methods A total of 117 blood samples were collected from 70 COVID-19 inpatients and convalescent patients. The presence of neutralizing antibody was determined with a modified cytopathogenic assay based on live SARS-CoV-2. The dynamics of neutralizing antibody levels at different with different clinical characteristics were analyzed. Results The seropositivity rate reached up to 100.0% within 20 days since onset, and remained 100.0% till day 41-53. The total GMT was 1:163.7 (95% CI, 128.5 to 208.6), and the antibody level was highest during day 31-40 since onset, and then decreased slightly. Individual differences in changes of antibody levels were observed among 8 representative convalescent patients. In multivariate GEE analysis, patients at age of 31-60 and 61-84 had a higher antibody level than those at age of 16-30 (β=1.0518, P =0.0152; β=1.3718, P =0.0020). Patients with a worse clinical classification had a higher antibody titer (β=0.4639, P =0.0227). Conclusions The neutralizing antibodies were detected even at the early stage of disease, and a significant response showed in convalescent patients. Moreover, changes on antibody levels ware individual specific.

It remains largely mysterious how the genomes of non-enveloped eukaryotic viruses are transferred across a membrane into the host cell. Picornaviruses are simple models for such viruses, and initiate this uncoating process through particle expansion, which reveals channels through which internal capsid proteins and the viral genome presumably exit the particle, although this has not been clearly seen until now. Here we present the atomic structure of an uncoating intermediate for the major human picornavirus pathogen CAV16, which reveals VP1 partly extruded from the capsid, poised to embed in the host membrane. Together with previous low-resolution results, we are able to propose a detailed hypothesis for the ordered egress of the internal proteins, using two distinct sets of channels through the capsid, and suggest a structural link to the condensed RNA within the particle, which may be involved in triggering RNA release. The detailed mechanism of how non-enveloped viruses initiate infection remains obscure. Ren et al. present the atomic structure of an uncoating intermediate for the human picornavirus CAV16, revealing a major capsid protein partly extruded from the capsid and suggesting a model for RNA release.

BACKGROUND: Emergence of severe acute respiratory syndrome (SARS) from the winter of 2002 to the spring of 2003 has caused a serious threat to public health. METHODS: To evaluate the safety and immunogenicity of the inactivated SARS coronavirus (SARS-CoV) vaccine, 36 subjects received two doses of 16 SARS-CoV units (SU) or 32 SU inactivated SARS-CoV vaccine, or placebo control. RESULTS: On day 42, the seroconversion reached 100% for both vaccine groups. On day 56, 100% of participants in the group receiving 16 SU and 91.1% in the group receiving 32 SU had seroconverted. The geometric mean titre of neutralizing antibody peaked 2 weeks after the second vaccination, but decreased 4 weeks later. CONCLUSION: The inactivated vaccine was safe and well tolerated and can elicit SARS-CoV-specific neutralizing antibodies.

BACKGROUND: The WHO declared COVID-19 a pandemic on March 11th, 2020. This serious outbreak and the precipitously increasing numbers of deaths worldwide necessitated the urgent need to develop an effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine. The development of COVID-19 vaccines has moved quickly. In this study, we assessed the efficacy, safety, and immunogenicity of an inactivated (SARS-CoV-2) vaccine. METHODS: We conducted a randomized, double-blind, placebo-controlled trial to evaluate the efficacy, immunogenicity, and safety of an inactivated SARS-CoV-2 vaccine and its lot-to-lot consistency. A total of 1620 healthy adults aged 18-59 years were randomly assigned to receive 2 injections of the trial vaccine or placebo on a day 0 and 14 schedule. This article was based on an interim report completed within 3 months following the last dose of study vaccine. The interim analysis includes safety and immunogenicity data for 540 participants in the immunogenicity subset and an efficacy analysis of the 1620 subjects. For the safety evaluation, solicited and unsolicited adverse events were collected after the first and second vaccination within 14 and 28 days, respectively. Blood samples were collected for an antibody assay before and 14 days following the second dose. RESULTS: Most of the adverse reactions were in the solicited category and were mild in severity. Pain at the injection site was the most frequently reported symptom. Antibody IgG titer determined by enzyme-linked immunosorbent assay was 97.48% for the seroconversion rate. Using a neutralization assay, the seroconversion rate was 87.15%. The efficacy in preventing symptomatic confirmed cases of COVID-19 occurring at least 14 days after the second dose of vaccine using an incidence rate was 65.30%. CONCLUSIONS: From the 3-month interim analysis, the vaccine exhibited a 65.30% efficacy at preventing COVID-19 illness with favorable safety and immunogenicity profiles.

The clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile tool for genomic engineering in mammalian cells and organisms, enabling the introduction of site-specific genomic double-strand breaks (DSBs) 1 . The resulting DSBs are repaired by at least two distinct and competitive mechanisms, nonhomologous end-joining (NHEJ) and homology-directed repair (HDR). The former results in insertions and deletions (indels), whereas the latter leads to precise genetic modification. Precise genetic modification is of great utility in both research and clinical applications, even though the frequency of HDR is inherently low 2 , 3 . Several strategies have recently been developed to enhance the efficiency of HDR, including genetic or chemical interruption of the NHEJ pathway 4 , 5 , overexpression of a positive HDR regulator 6 and manipulation of the cell cycle 7 . For example, Scr7, a DNA ligase IV inhibitor, has been shown to enhance HDR efficiency up to 2-4-fold in CRISPR/Cas9-manipulated embryos 4 . However, interference with endogenous genes has potentially detrimental side effects. Furthermore, such strategies are best suited to the knock-in of a short DNA fragment, normally no larger than 30 bp ( Supplementary information, Figure S1A ). The precise knock-in of a larger DNA fragment, such as one as large as 1 000 bp, is still a challenging task in mice 8 . Even for a donor DNA with ∼ 1-8 kb homology arms, the knock-in efficiency achieved by CRISPR/Cas9 was lower than 6% in mice 9 , 10 , 11 , 12 . The Cas9 protein preferentially remains at the targeting site, where it first releases the non-target strand and could anchor there for as long as 6 h 13 . We hypothesized that the donor DNA searching for a homologous sequence across the entire genome is a key rate-limiting step of HDR, and thus the donor DNA would have a greater chance of being used if it is associated with CRISPR/Cas9 components. Here we demonstrate a simple and robust method of enhancing the HDR efficiency with no side effects detected. The newly developed system, termed the Cas9-Avidin-Biotin ssDNA (CAB) system, achieved an HDR frequency of ∼ 20% in mouse embryos for insertions of ∼ 1 kb. The CAB system is composed of a Cas9 fused with avidin via a flexible linker, an sgRNA and a biotin-modified single-strand donor DNA (biotin-ssDNA) ( Figure 1A ). Due to the high affinity between avidin and biotin, biotin-ssDNA would be enriched at the cleavage site, a hypothesis which was supported by the membrane labeling experiment ( Supplementary information, Figure S1B ). First, we optimized the CAB system, and found that Cas9 fused at its C-terminus to avidin still retains activity similar to wild-type Cas9 and the linker length should be of at least 16 amino acids ( Supplementary information, Figure S1C ). These results suggest that an intact Cas9 N-terminus is essential for nuclease activity, which is consistent with a previous structural analysis of Cas9 14 . Next, we assessed the performance of the optimized CAB system in 293T cells. The reporter assay showed a 2-fold enhancement of HDR efficiency, whereas sequencing analysis of editing at an endogenous gene locus revealed an elevation of the precise editing frequency by ∼ 5-fold ( Supplementary information, Figure S1D-S1F ). The CAB system also increased the ratio of the precise knock-in of a short DNA fragment up to 3-fold compared with the traditional CRISPR/Cas9 system in mouse zygotes ( Supplementary information, Figure S2A-S2C ). Figure 1 The CAB system enables precise insertion of double floxp sites with high efficiency. (A) Schematic diagram showing the CAB system. Above: a single-strand donor DNA is modified by biotin at the 5′-terminus and it contains an insertion sequence (blue line) flanked by homology arms. Guided by sgRNA, the Cas9-avidin fusion protein induces DSB at the desired locus and enriches the donor DNA. Bottom: strategy for inducing the double-floxp insertion in a targeted gene locus. A single-strand DNA donor contains two floxp sites, each with a homology arm of ∼ 100 nt at one side. Genome amplification primers (red arrows) are located outside the donor region. Two sets of loxp detection primers are located near the upstream/downstream floxp sites, respectively. Once a floxp site is successfully inserted, a larger band appears above the wild-type band, termed the knock-in band. (B) Summary of CAB-mediated HDR in four genes including PRKACA , Sirt7 , ARF6 and UQCC3 . The control results using the wild-type CRISPR/Cas9 system are shown at the bottom. (C) Genome analyses of the F1 PRKACA mice. Left: scheme of the process and validation of germline transmission. Middle: upstream/downstream loxp insertion was detected using detection primer sets. Right: the complete sequence of the knock-in allele was analyzed by sequencing using amplification primers, and a representative sequence of a positive F1 is shown. (D) HDR-mediated knock-in of double floxp sites in the indicated loci. Genomic DNA was extracted from the tail of F0 pups and amplified by PCR using the detection primer sets to identify the loxp insertion. The ratio of the precise knock-in allele for each positive F0 pup was quantified according to the intensities of the knock-in band and wild-type band as shown in Supplementary information, Figure S2G-S2I . (E) Genotype analysis of F1 pups in all of the four genetic types of mice. In each type, one founder mouse, i.e., No. 6 in Sirt7 , No. 10 in UQCC3 , No. 1 in PRKACA and No. 13 in ARF6 , was selected to cross with a wild-type mouse, and genomic DNA of F1 mice was then analyzed by PCR. Of note, all positive F1 mice carried one knock-in allele and one wild-type allele. Full size image The Cre/loxp system has been widely used to generate conditional knockout mice. Thus, we investigated whether the CAB system can achieve the knock-in of double floxp sites in one step. The CAB or CRISPR/Cas9 components were microinjected into a fertilized mouse zygote. The manipulated embryos were cultured to the two-cell stage and transferred into pseudopregnant females. The tails of the pups carried to term were harvested for PCR and sequencing analyses. PRKACA , the gene for the catalytic subunit α of protein kinase A, has been linked to Cushing's syndrome, adrenocortical tumors, hepatocellular carcinoma and other diseases due to mutation or abnormal fusion. We sought to generate mice carrying two floxp sites inserted in the fifth and eighth introns of PRKACA , respectively ( Supplementary information, Figure S2D ). Two cleavage sites were selected according to the cutting efficiency of the CRISPR/Cas9 system, and a ∼ 1 000-nt donor DNA flanked by 100-nt homology arms was synthesized. 50% of the embryos manipulated by the CAB system generated live pups, demonstrating a survival rate comparable to that yielded by the CRISPR/Cas9 system ( Figure 1B ). At 7 days of age, DNA extracted from the pup tails was analyzed by PCR using two detection primer sets and sequencing using amplification primers. No precise editing was observed for the CRISPR/Cas9 system, and 36% of mice (4 out of 11) exhibited indels, indicating that DSBs were repaired via NHEJ ( Figure 1B ). In the CAB group, ∼ 15% pups (2 out of 14, i.e., No. 1 and No. 5) exhibited precise knock-in, which was confirmed by DNA sequencing ( Figure 1B and Supplementary information, Figure S2E ). The percentages of the precise knock-in alleles in the No. 1 and No. 5 mice were approximately 25% and 2%, respectively ( Figure 1D ). To demonstrate the successful germline transmission, the No. 1 founder mouse was crossed with a wild-type mouse, and five of six offspring were shown to carry the knock-in allele ( Figure 1C and Supplementary information, Figure S2F ). To further demonstrate the robustness of the CAB system, we generated three additional mouse genetic models carrying double floxp sites at UQCC3 , ARF6 or Sirt7 gene ( Figure 1B and Supplementary information, Figure S2D ). Genotyping performed 7 days after the pup birth showed that 18%-22% of the F0 mice carried the knock-in alleles ( Figure 1B ), suggesting that the improvement in precise editing obtained using the CAB system is not limited to a particular locus. It is noteworthy that a large proportion of the positive F0 pups exhibited a ratio of over 50% for the precise knock-in allele ( Figure 1D and Supplementary information, Figure S2G-S2I ). Particularly in case of the ARF6 locus, the No. 13 F0 pup is likely homozygous for the knock-in allele as PCR analysis only revealed the knock-in band ( Figure 1D and Supplementary information, Figure S2G ) and all 14 TA clones sequenced were positive for double floxp sites (data not shown). Finally, we selected one F0 founder from each genetic type and crossed them with wild-type mice for germline transmission analysis. All four F0 founder mice produced F1 pups carrying the knock-in allele. Of note, 60%-100% of the F1 pups of each genotype harbored the knock-in allele ( Figure 1E ), suggesting that a large proportion of germ cells of the four F0 founders carried the knock-in allele. Thus, it is likely feasible to generate a heterozygous conditional knockout mouse within 6 months after obtaining the F0 founders by crossing the F0 mice harboring high levels of knock-in mosaicism with a Cre mouse. Here we report a simple and robust system to enhance HDR-mediated precise genomic editing in mouse embryos via the enrichment of donor DNA at the cleavage sites. Over 5 000 genetic diseases exist in humans; however, less than 10% have been mechanistically characterized and no gene editing therapy has been approved by FDA for clinical use. CRISPR/Cas9 is a powerful tool for gene modification in a variety of organisms as well as human cells 1 . A large number of challenging issues need to be addressed before this powerful technology can come into clinical use, including the low efficiency of gene correction based on HDR. Using the CAB system, we have improved the HDR efficiency such that HDR-based precise editing occurred in ∼ 20% of the manipulated embryos. In summary, this system has three advantages. First, the donor DNA requires only two short homology arms of < 100 nt and thus can be easily synthesized. This is in contrast with traditional CRISPR/Cas9-mediated precise editing which requires long homology arms of a few kbs 9 , 10 , 11 , 12 . Second, this system does not cause additional toxicity compared with the CRISPR/Cas9 system, as evidenced by a similar survival rate of the F0 pups in embryos manipulated by these two systems. Safety is a critical concern for any CRISPR system in terms of future clinical application. Although chemical or genetic interruption of the NHEJ pathway can favor HDR, such manipulation may have a detrimental effect on embryonic development. It has been reported that Scr7 treatment results in defects in lymphocyte development 15 . Finally, we observed that 60%-100% of the F1 pups carried the knock-in allele with double floxp sites, suggesting that the new system would enhance the efficiency of generating a mouse model with precise genetic modification and thus save time compared with conventional approaches.

Tumor tissues/cells are the best sources of antigens to prepare cancer vaccines. However, due to the difficulty of solubilization and delivery of water-insoluble antigens in tumor tissues/cells, including water-insoluble antigens into cancer vaccines and delivering such vaccines efficiently to antigen-presenting cells (APCs) remain challenging. To solve these problems, herein, water-insoluble components of tumor tissues/cells are solubilized by 8 m urea and thus whole components of micrometer-sized tumor cells are reasssembled into nanosized nanovaccines. To induce maximized immunization efficacy, various antigens are loaded both inside and on the surface of nanovaccines. By encapsulating both water-insoluble and water-soluble components of tumor tissues/cells into nanovaccines, the nanovaccines are efficiently phagocytosed by APCs and showed better therapeutic efficacy than the nanovaccine loaded with only water-soluble components in melanoma and breast cancer. Anti-PD-1 antibody and metformin can improve the efficacy of nanovaccines. In addition, the nanovaccines can prevent lung cancer (100%) and melanoma (70%) efficiently in mice. T cell analysis and tumor microenvironment analysis indicate that tumor-specific T cells are induced by nanovaccines and both adaptive and innate immune responses against cancer cells are activated by nanovaccines. Overall, this study demonstrates a universal method to make tumor-cell-based nanovaccines for cancer immunotherapy and prevention.

The gut microbiota is increasingly recognized to influence brain function through the gut-brain axis.Albiflorin, an antidepressant natural drug in China with a good safety profile, is difficult to absorb and cannot be detected in the brain after oral administration.Accordingly, the antidepressant mechanism of albiflorin in vivo has not been elucidated clearly.Methods: We identified benzoic acid as the characteristic metabolite of albiflorin in vivo and in vitro, then discovered the roles of gut microbiota in the conversion of albiflorin by carboxylesterase.Pharmacodynamic and pharmacokinetic studies were performed for the antidepressant activities of albiflorin in animals, and the efficacy of benzoic acid in inhibiting D-amino acid oxidase (DAAO) in brain was further investigated.Results: We validated that gut microbiota transformed albiflorin to benzoic acid, a key metabolite in the intestine that could cross the blood-brain barrier and, as an inhibitor of DAAO in the brain, improved brain function and exerted antidepressant activity in vivo.Intestinal carboxylesterase was the crucial enzyme that generated benzoic acid from albiflorin.Additionally, the regulatory effect of albiflorin on the gut microbiota composition was beneficial to alleviate depression.Conclusion: Our findings suggest a novel gut-brain dialogue through intestinal benzoic acid for the treatment of depression and reveal that the gut microbiota may play a causal role in the pathogenesis and treatment of the central nervous system disease.

PURPOSE: Targeting the protein neddylation pathway has become an attractive anticancer strategy; however, the role of death receptor-mediated extrinsic apoptosis during treatment remained to be determined. EXPERIMENTAL DESIGN: The activation of extrinsic apoptosis and its role in MLN4924 treatment of human esophageal squamous cell carcinoma (ESCC) were evaluated both in vitro and in vivo The expression of the components of extrinsic apoptotic pathway was determined by immunoblotting analysis and downregulated by siRNA silencing for mechanistic studies. RESULTS: Pharmaceutical or genetic inactivation of neddylation pathway induced death receptor 5 (DR5)-mediated apoptosis and led to the suppression of ESCC in murine models. Mechanistically, neddylation inhibition stabilized activating transcription factor 4 (ATF4), a Cullin-Ring E3 ubiquitin ligases (CRL) substrate. Transcription factor CHOP was subsequently transactivated by ATF4 and further induced the expression of DR5 to activate caspase-8 and induce extrinsic apoptosis. Moreover, the entire neddylation pathway was hyperactivated in ESCC and was negatively associated with patient overall survival. CONCLUSIONS: Our findings highlight a critical role of ATF4-CHOP-DR5 axis-mediated extrinsic apoptosis in neddylation-targeted cancer therapy and support the clinical investigation of neddylation inhibitors (e.g., MLN4924) for the treatment of ESCC, a currently treatment-resistant disease with neddylation hyperactivation. Clin Cancer Res; 22(16); 4145-57. ©2016 AACR.

Structurally and genetically, human herpesviruses are among the largest and most complex of viruses. Using cryo-electron microscopy (cryo-EM) with an optimized image reconstruction strategy, we report the herpes simplex virus type 2 (HSV-2) capsid structure at 3.1 angstroms, which is built up of about 3000 proteins organized into three types of hexons (central, peripentonal, and edge), pentons, and triplexes. Both hexons and pentons contain the major capsid protein, VP5; hexons also contain a small capsid protein, VP26; and triplexes comprise VP23 and VP19C. Acting as core organizers, VP5 proteins form extensive intermolecular networks, involving multiple disulfide bonds (about 1500 in total) and noncovalent interactions, with VP26 proteins and triplexes that underpin capsid stability and assembly. Conformational adaptations of these proteins induced by their microenvironments lead to 46 different conformers that assemble into a massive quasisymmetric shell, exemplifying the structural and functional complexity of HSV.