A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS.

Vaccines are urgently needed to control the ongoing pandemic COVID-19 and previously emerging MERS/SARS caused by coronavirus (CoV) infections. The CoV spike receptor-binding domain (RBD) is an attractive vaccine target but is undermined by limited immunogenicity. We describe a dimeric form of MERS-CoV RBD that overcomes this limitation. The RBD-dimer significantly increased neutralizing antibody (NAb) titers compared to conventional monomeric form and protected mice against MERS-CoV infection. Crystal structure showed RBD-dimer fully exposed dual receptor-binding motifs, the major target for NAbs. Structure-guided design further yielded a stable version of RBD-dimer as a tandem repeat single-chain (RBD-sc-dimer) which retained the vaccine potency. We generalized this strategy to design vaccines against COVID-19 and SARS, achieving 10- to 100-fold enhancement of NAb titers. RBD-sc-dimers in pilot scale production yielded high yields, supporting their scalability for further clinical development. The framework of immunogen design can be universally applied to other beta-CoV vaccines to counter emerging threats.

Automated Droplet Ejection from a Digital Microfluidics Sample Pretreatment Device Enables Batch-Mode Chemiluminescence Immunoassay.

Digital microfluidics (DMF) features programmed manipulation of fluids in multiple steps, making it a valuable tool for sample pretreatment. However, the integration of sample pretreatment with its downstream reaction and detection requires transferring droplets from the DMF device to the outside world. To address this issue, the present study developed a modified DMF device that allows automated droplet ejection out of the chip, facilitated by a tailor-designed interface. A double-layered DMF microchip with an oil-filled medium was flipped over, with a liquid infusion port and a liquid expulsion port accommodated on the top working PCB plate and the bottom grounded ITO plate, respectively, to facilitate chip-to-world delivery of droplets. Using chemiluminescent immunoassay (CLIA) as an illustrative application, the sample pretreatment was programmed on the DMF device, and CLIA droplets were ejected from the chip for signal reading. In our workflow, CLIA droplets can be ejected from the DMF device through the chip-to-world interface, freeing up otherwise occupied electrodes for more sample pretreatment and enabling streamlined droplet microreactions and batch-mode operation for bioanalysis. Integrated with these interfacing portals, the DMF system achieved a single-channel throughput of 17 samples per hour, which can be further upscaled for more productive applications by parallelizing the DMF modules. The results of this study demonstrate that the droplet ejection function that is innovated in a DMF sample pretreatment microsystem can significantly improve analytical throughput, providing an approach to establishing an automated but decentralized biochemical sample preparation workstation for large-scale and continuous bioanalysis.

Rapid Detection of Uropathogens Using an Integrated Multiplex Digital Nucleic Acid Detection Assay Powered by a Digital-to-Droplet Microfluidic Device.

The digital nucleic acid detection assay features the capability of absolute quantitation without the need for calibration, thereby facilitating the rapid identification of pathogens. Although several integrated digital nucleic acid detection techniques have been developed, there are still constraints in terms of automation and analysis throughput. To tackle these challenges, this study presents a digital-to-droplet microfluidic device comprising a digital microfluidics (DMF) module at the bottom and a droplet microfluidics module at the top. Following sample introduction, the extraction of nucleic acid and the dispensation of nucleic acid elution for mixing with the multiple amplification reagents are carried out in the DMF module. Subsequently, the reaction droplets are transported to the sample inlet of the droplet microfluidic module via a liquid outlet, and then droplet generation in four parallel units within the droplet microfluidics module is actuated by negative pressure generated by a syringe vacuum. The digital-to-droplet microfluidic device was employed to execute an integrated multiplex digital droplet nucleic acid detection assay (imDDNA) incorporating loop-mediated isothermal amplification (LAMP). This assay was specifically designed to enable simultaneous detection of four uropathogens, namely, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterococcus faecalis. The entire process of the imDDNA is completed within 75 min, with a detection range spanning 5 orders of magnitude (9.43 × 10-2.86 × 104 copies µL-1). The imDDNA was employed for the detection of batched clinical specimens, showing a consistency of 91.1% when compared with that of the conventional method. The imDDNA exhibits simplicity in operation and accuracy in quantification, thus offering potential advantages in achieving rapid pathogen detection.

A fully automated microfluidic PCR-array system for rapid detection of multiple respiratory tract infection pathogens.

Rapid and accurate identification of respiratory tract infection pathogens is of utmost importance for clinical diagnosis and treatment, as well as prevention of pathogen transmission. To meet this demand, a microfluidic chip-based PCR-array system, Onestart, was developed. The Onestart system uses a microfluidic chip packaged with all the reagents required, and the waste liquid is also collected and stored on the chip. This ready-to-use system can complete the detection of 21 pathogens in a fully integrated manner, with sample lysis, nucleic acid extraction/purification, and real-time PCR sequentially implemented on the same chip. The entire analysis process is completed within 1.5 h, and the system automatically generates a test report. The lower limit-of-detection (LOD) of the Onestart assay was determined to be 1.0 × 103 copies·mL-1. The inter-batch variation of cycle threshold (Ct) values ranged from 0.08% to 0.69%, and the intra-batch variation ranged from 0.9% to 2.66%. Analytical results of the reference sample mix showed a 100% specificity of the Onestart assay. The analysis of batched clinical samples showed consistency of the Onestart assay with real-time PCR. With its ability to provide rapid, sensitive, and specific detection of respiratory tract infection pathogens, application of the Onestart system will facilitate timely clinical management of respiratory tract infections and effective prevention of pathogen transmission. Onestart, a ready-to-use system, can detect 21 pathogens in a fully integrated manner on a microchip within 1.5 h.

Droplet Encoding-Pairing Enabled Multiplexed Digital Loop-Mediated Isothermal Amplification for Simultaneous Quantitative Detection of Multiple Pathogens.

Despite the advantages of digital nucleic acid analysis (DNAA) in terms of sensitivity, precision, and resolution, current DNAA methods commonly suffer a limitation in multiplexing capacity. To address this issue, a droplet encoding-pairing enabled DNAA multiplexing strategy is developed, wherein unique tricolor combinations are deployed to index individual primer droplets. The template droplets and primer droplets are sequentially introduced into a microfluidic chip with a calabash-shaped microwell array and are pairwise trapped and merged in the microwells. Pre-merging and post-amplification image analysis with a machine learning algorithm is used to identify, enumerate, and address the droplets. By incorporating the amplification signals with droplet encoding information, simultaneous quantitative detection of multiple targets is achieved. This strategy allows for the establishment of flexible multiplexed DNAA by simply adjusting the primer droplet library. Its flexibility is demonstrated by establishing two multiplexed (8-plex) droplet digital loop-mediated isothermal amplification (mddLAMP) assays for individually detecting lower respiratory tract infection and urinary tract infection causative pathogens. Clinical sample analysis shows that the microbial detection outcomes of the mddLAMP assays are consistent with those of the conventional assay. This DNAA multiplexing strategy can achieve flexible high-order multiplexing on demand, making it a desirable tool for high-content pathogen detection.

Developmental and reproductive toxicity of a recombinant protein subunit COVID-19 vaccine (ZF2001) in rats.

ZF2001, a protein subunit vaccine against coronavirus disease 2019 (COVID-19), contains recombinant tandem repeat of dimeric receptor-binding domain (RBD) protein of the SARS-CoV-2 spike protein with an aluminium-based adjuvant. During the development of this vaccine, two nonclinical studies were conducted to evaluate female fertility, embryo-fetal development, and postnatal developmental toxicity in Sprague‒Dawley rats according to the ICH S5 (R3) guideline. In Study 1 (embryo-fetal developmental toxicity, EFD), 144 virgin female rats were randomly assigned into four groups and received three doses of vaccine (25 µg or 50 µg RBD protein/dose, containing the aluminium-based adjuvant), the aluminium-based adjuvant or a sodium chloride injection administered intramuscularly on days 21 and 7 prior to mating and on gestation day (GD) 6. In Study 2 (pre- and postnatal developmental toxicity, PPND), ZF2001 at a dose of 25 µg RBD protein/dose or sodium chloride injection was administered intramuscularly to female rats (n = 28 per group) 7 days prior to mating and on GD 6, GD 20 and postnatal day (PND) 10. There were no obvious adverse effects in dams, except for local injection site reactions related to the aluminium-based adjuvant (yellow nodular deposits in the interstitial muscle fibres). There were also no effects of ZF2001 on the mating performance, fertility or reproductive performance of parental females, embryo-fetal development, postnatal survival, growth, physical development, reflex ontogeny, behavioural and neurofunctional development, or reproductive performance of the offspring. The strong immune responses associated with binding and neutralising antibodies were both confirmed in dams and fetuses or offspring in these two studies. These results would support clinical trials or the use of ZF2001 in maternal immunisation campaigns, including those involving women with childbearing potential, regardless of pregnancy status.

Active droplet-array microfluidics-based chemiluminescence immunoassay for point-of-care detection of procalcitonin.

The application of conventional chemiluminescence immunoassay (CLIA) in resource-limited settings is limited due to the large apparatus footprint, cumbersome operation and maintenance process, and high consumption of reagents. To address this issue, we developed an active droplet-array (ADA) microfluidics-based CLIA system, which consists of a compact microchip analyzer and microfluidic chips with preloaded reagents. The microfluidic chip contains microslit-connected microchambers, in which all the required reagents were preloaded in water-in-oil droplets. The microfluidic chip analyzer can manipulate five microfluidic chips in parallel in a single run. By interacting the microchip with magnetic, thermal, optical mechanisms programmatically, the entire workflow of CLIA can be accomplished in an automated manner. With the proposed CLIA, the detection of procalcitonin (PCT) can be completed in 12 min, with a limit of detection (LOD) of 0.044 ng mL-1 and a detection range from 0.044 to 100 ng mL-1. We found a good linear correlation between the microfluidic CLIA and the conventional electrochemiluminescence immunoassay (R2=0.98).The microfluidic CLIA has significant advantages over the conventional ELISA in detection sensitivity, dynamic range, instrument size and turnaround time, and can provide more consistent and reliable results than the lateral flow immunoassays. The compact microfluidic system can perform automated and parallelized CLIA in a short turnaround time, and thus well suited to Point-of-Care detection of disease biomarkers.

Preclinical Toxicity and Immunogenicity of a COVID-19 Vaccine (ZF2001) in Cynomolgus Monkeys.

Although the new coronavirus disease 2019 (COVID-19) outbreak occurred in late 2019, it is still endemic worldwide, and has become a global public health problem. Vaccination against SARS-CoV-2 is considered to be the most effective intervention to prevent the spread of COVID-19. ZF2001 is a recombinant protein vaccine based on SARS-CoV-2 receptor-binding domain (RBD) subunit which contains aluminum adjuvant. In order to advance our research on ZF2001 into clinical trial, we investigated the general toxicity and immunogenicity of ZF2001 in cynomolgus monkeys and assessed the possible target organs for vaccine-induced toxicity. In the present research, we observed no significant systemic toxicities and abnormal cardiovascular and respiratory events following four times injections of intramuscular ZF2001 in cynomolgus monkeys. Histological examination revealed recoverable inflammatory changes in quadricep muscle and adjacent lymph node at the vaccine injection site. As expected, the vaccine can produce a strongly specific binding antibody and neutralizing antibodies in cynomolgus monkeys after inoculation. Taken together, our regulatory toxicology research proves the safety and immunogenicity of the ZF2001 vaccine, supporting its entry into large scale clinical trials.

A tandem-repeat dimeric RBD protein-based covid-19 vaccine zf2001 protects mice and nonhuman primates.

Safe, efficacious, and deployable vaccines are urgently needed to control COVID-19 in the large-scale vaccination campaigns. We report here the preclinical studies of an approved protein subunit vaccine against COVID-19, ZF2001, which contains tandem-repeat dimeric receptor-binding domain (RBD) protein with alum-based adjuvant. We assessed vaccine immunogenicity and efficacy in both mice and non-human primates (NHPs). ZF2001 induced high levels of RBD-binding and SARS-CoV-2 neutralizing antibody in both mice and non-human primates, and elicited balanced TH1/TH2 cellular responses in NHPs. Two doses of ZF2001 protected Ad-hACE2-transduced mice against SARS-CoV-2 infection, as detected by reduced viral RNA and relieved lung injuries. In NHPs, vaccination of either 25 µg or 50 µg ZF2001 prevented infection with SARS-CoV-2 in lung, trachea, and bronchi, with milder lung lesions. No evidence of disease enhancement was observed in both animal models. ZF2001 has been approved for emergency use in China, Uzbekistan, Indonesia, and Columbia. The high safety, immunogenicity, and protection efficacy in both mice and NHPs found in this preclinical study was consistent with the results in human clinical trials.

A pocket-sized device automates multiplexed point-of-care RNA testing for rapid screening of infectious pathogens.

Rapid screening of infectious pathogens at the point-of-care (POC) is ideally low-cost, portable, easy to use, and capable of multiplex detection with high sensitivity. However, satisfying all these features in a single device without compromise remains a challenging task. Here, we introduce an ultraportable, automated RNA amplification testing device that allows rapid screening of infectious pathogens from clinical samples. In this device, 3D-printed structural parts incorporated with off-the-shelf mechanic/electronic components are utilized to create an inexpensive and automated droplet manipulation platform. On this platform, a simple configuration that couples a linear displacement of the chip with a tunable magnet array allows parallel and versatile droplet operations, including mixing, splitting, transporting, and merging. By exploiting a multi-channel droplet array chip to preload necessary reagents in "water-in-oil" format, bacteria lysis, RNA extraction and amplification are seamlessly integrated and implemented by the combination of droplet operations. Furthermore, visual readout and geometrically-multiplexed quantitative detection are provided by an integrated wireless video camera-enabled wide-field fluorescence imaging. We demonstrated that this droplet-based device could have a shorter RNA extraction time (12 min) and lower detection limits for pathogenic RNA (approaching to 102 copies per reaction). We also verified its clinical applicability for the rapid screening of four sexually transmitted pathogens from urine specimens. Results show that the sample-to-answer assay could be completed in approximately 42 min, with 100% concordance with the laboratory-based molecular testing. The exhibiting features may render this microdevice an easily accessible POC molecular diagnostic platform for infectious disease, especially in resource-limited settings.

Safety and immunogenicity of a recombinant tandem-repeat dimeric RBD-based protein subunit vaccine (ZF2001) against COVID-19 in adults: two randomised, double-blind, placebo-controlled, phase 1 and 2 trials.

BACKGROUND: Although several COVID-19 vaccines have been developed so far, they will not be sufficient to meet the global demand. Development of a wider range of vaccines, with different mechanisms of action, could help control the spread of SARS-CoV-2 globally. We developed a protein subunit vaccine against COVID-19 using a dimeric form of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein as the antigen. We aimed to assess the safety and immunogenicity of this vaccine, ZF2001, and determine the appropriate dose and schedule for an efficacy study. METHODS: We did two randomised, double-blind, placebo-controlled, phase 1 and phase 2 trials. Phase 1 was done at two university hospitals in Chongqing and Beijing, China, and phase 2 was done at the Hunan Provincial Center for Disease Control and Prevention in Xiangtan, China. Healthy adults aged 18-59 years, without a history of SARS-CoV or SARS-CoV-2 infection, an RT-PCR-positive test result for SARS-CoV-2, a history of contact with confirmed or suspected COVID-19 cases, and severe allergies to any component of the vaccine were eligible for enrolment. In phase 1, participants were randomly assigned (2:2:1) to receive three doses of the vaccine (25 µg or 50 µg) or placebo intramuscularly, 30 days apart. In phase 2, participants were randomly assigned (1:1:1:1:1:1) to receive the vaccine (25 µg or 50 µg) or placebo intramuscularly, 30 days apart, in either a two-dose schedule or a three-dose schedule. Investigators, participants, and the laboratory team were masked to group allocation. For phase 1, the primary outcome was safety, measured by the occurrence of adverse events and serious adverse events. For phase 2, the primary outcome was safety and immunogenicity (the seroconversion rate and the magnitude, in geometric mean titres [GMTs], of SARS-CoV-2-neutralising antibodies). Analyses were done on an intention-to-treat and per-protocol basis. These trials are registered with ClinicalTrials.gov (NCT04445194 and NCT04466085) and participant follow-up is ongoing. FINDINGS: Between June 22 and July 3, 2020, 50 participants were enrolled into the phase 1 trial and randomly assigned to receive three doses of placebo (n=10), the 25 µg vaccine (n=20), or the 50 µg vaccine (n=20). The mean age of participants was 32·6 (SD 9·4) years. Between July 12 and July 17, 2020, 900 participants were enrolled into the phase 2 trial and randomly assigned to receive two doses of placebo (n=150), 25 µg vaccine (n=150), or 50 µg vaccine (n=150), or three doses of placebo (n=150), 25 µg vaccine (n=150), or 50 µg vaccine (n=150). The mean age of participants was 43·5 (SD 9·2) years. In both phase 1 and phase 2, adverse events reported within 30 days after vaccination were mild or moderate (grade 1 or 2) in most cases (phase 1: six [60%] of ten participants in the placebo group, 14 [70%] of 20 in the 25 µg group, and 18 [90%] of 20 in the 50 µg group; phase 2: 37 [25%] of 150 in the two-dose placebo group, 43 [29%] of 150 in the two-dose 25 µg group, 50 [33%] of 150 in the two-dose 50 µg group, 47 [31%] of 150 in the three-dose placebo group, 72 [48%] of 150 in the three-dose 25 µg group, and 65 [43%] of 150 in the three-dose 50 µg group). In phase 1, two (10%) grade 3 or worse adverse events were reported in the 50 µg group. In phase 2, grade 3 or worse adverse events were reported by 18 participants (four [3%] in the two-dose 25 µg vaccine group, two [1%] in the two-dose 50 µg vaccine group, two [1%] in the three-dose placebo group, four [3%] in the three-dose 25 µg vaccine group, and six [4%] in the three-dose 50 µg vaccine group), and 11 were considered vaccine related (two [1%] in the two-dose 25 µg vaccine group, one [1%] in the two-dose 50 µg vaccine group, one [1%] in the three-dose placebo group, two [1%] in the three-dose 25 µg vaccine group, and five [3%] in the three-dose 50 µg vaccine group); seven participants reported serious adverse events (one [1%] in the two-dose 25 µg vaccine group, one [1%] in the two-dose 50 µg vaccine group, two [1%] in the three-dose placebo group, one [1%] in the three-dose 25 µg vaccine group, and two [1%] in the three-dose 50 µg vaccine group), but none was considered vaccine related. In phase 2, on the two-dose schedule, seroconversion rates of neutralising antibodies 14 days after the second dose were 76% (114 of 150 participants) in the 25 µg group and 72% (108 of 150) in the 50 µg group; on the three-dose schedule, seroconversion rates of neutralising antibodies 14 days after the third dose were 97% (143 of 148 participants) in the 25 µg group and 93% (138 of 148) in the 50 µg group. In the two-dose groups in phase 2, the SARS-CoV-2-neutralising GMTs 14 days after the second dose were 17·7 (95% CI 13·6-23·1) in the 25 µg group and 14·1 (10·8-18·3) in the 50 µg group. In the three-dose groups in phase 2, the SARS-CoV-2-neutralising GMTs 14 days after the third dose were 102·5 (95% CI 81·8-128·5) in the 25 µg group and 69·1 (53·0-90·0) in the 50 µg group. INTERPRETATION: The protein subunit vaccine ZF2001 appears to be well tolerated and immunogenic. The safety and immunogenicity data from the phase 1 and 2 trials support the use of the 25 µg dose in a three-dose schedule in an ongoing phase 3 trial for large-scale evaluation of ZF2001's safety and efficacy. FUNDING: National Program on Key Research Project of China, National Science and Technology Major Projects of Drug Discovery, Strategic Priority Research Program of the Chinese Academy of Sciences, and Anhui Zhifei Longcom Biopharmaceutical. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Enzymatic characterization of a monomeric isocitrate dehydrogenase from Streptomyces lividans TK54.

Isocitrate dehydrogenase (IDH) is one of the key enzymes in the citric acid cycle, which involves in providing energy and biosynthetic precursors for metabolism. Here, we report for the first time the enzymatic characterization of a monomeric NADP(+)-dependent IDH from Streptomyces lividans TK54 (SlIDH). The icd gene (GenBank database accession number EU661252) encoding IDH was cloned and overexpressed in Escherichia coli. The molecular mass of SlIDH was about 80 kDa, typical of a monomeric NADP-IDH, and showed high amino acid sequence identity with known monomeric IDHs. The optimal activity of the 6His-tagged SlIDH was found at pH values 8.5 (Mn(2+)) and 9.0 (Mg(2+)), and the optimal temperature was around 46 degrees C. Heat-inactivation studies showed that about 50% SlIDH activity was preserved at 38 degrees C after 20 min of incubation. The recombinant SlIDH displayed a 62,000-fold (k(cat)/K(m)) preference for NADP(+) over NAD(+) with Mn(2+), and a 85,000-fold greater specificity for NADP(+) than NAD(+) with Mg(2+). Therefore, SlIDH is a divalent cation-dependent monomeric IDH with remarkably high coenzyme preference for NADP(+).