Human induced pluripotent stem cells (hiPSCs) may acquire genomic alterations during reprogramming and culture, which poses significant risks for clinical applications. Current detection methods, such as karyotyping analysis, often fail to identify critical submicroscopic variations. This highlights an urgent need for comprehensive genomic surveillance strategies. Three human iPSC lines were continually cultured in vitro for 50 passages, with genome alterations evaluated every 10 passages. The evaluation methods included karyotyping to detect chromosomal abnormalities, optical genome mapping (OGM) to identify copy number variations (CNVs) and structural variants (SVs), whole-exome sequencing (WES) to detect coding mutations, and RNA sequencing (RNA-seq) to detect the changes of gene expression. We detected accumulating chromosomal abnormalities (e.g., trisomy 12), SVs, CNVs, and sequence mutations in three hiPSC lines during extended culture. OGM effectively identified SVs and CNVs below karyotyping resolution, particularly recurrent genome abnormalities such as gains on chr17q, chr12p and chr20q. WES revealed coding mutations, including germline short variants and newly acquired somatic mutations, some of which were associated with tumors or diseases, such as CDH1, BCOR. Transcriptional changes correlated with genomic alterations, including dysregulation of oncogenes such as BCL2L1, KRAS and MDM2. Results demonstrate that each method had unique detection capabilities and limitations, and only integrative approaches can comprehensively identify genomic abnormalities. This study established a comprehensive strategy for evaluating the genomic alterations of hiPSCs by integrating karyotyping, OGM, WES, and RNA-seq. This comprehensive strategy can be applied to scenarios such as hiPSC clone screening, establishment of cell bank passages, and quality control of hiPSC-derived products. It provides a reliable genetic stability evaluation protocol to support the safe clinical application of hiPSC-related products.

Background: Mesenchymal stem/stromal cells (MSCs) are increasingly applied in the treatment of graft-versus-host disease, autoimmune disorders, and severe inflammatory conditions, frequently in patients with impaired immune function, where viral safety is a critical concern. Human herpesvirus 6 (HHV-6) is distinctive in that it is widespread in the general population and has been implicated in disorders that overlap with common MSC therapeutic indications, where immunosuppression or inflammation may facilitate HHV6 reactivation. Yet, its presence in MSC products has not been reported. Methods: Clinical-grade MSC batches were initially screened for HHV6 by qPCR. The positive sample was further analyzed by metagenomic next-generation sequencing (mNGS). Viral load was quantified by droplet digital PCR (ddPCR), and its integration sites was investigated with PacBio HiFi long-reads sequencing.. Results: HHV-6 DNA was detected in an umbilical cord–derived MSC batch (MSC-1090) at passages 3, 5, and 10. mNGS identified 100–144 HHV-6B–specific reads, covering 16.44–18.89% of the viral genome, without detectable viral RNA. ddPCR demonstrated approximately one copy of HHV-6 DNA per cell across passages, indicating stable persistence. Long-read sequencing and genomic alignment suggested integration of HHV-6B into the telomeric region of chromosome 19. Conclusions This study reports the first detection of HHV-6B DNA in a clinical-grade MSC product, revealing a previously unrecognized gap in risk-based viral safety assessment and suggesting that HHV-6 may warrant consideration in MSC quality control strategies.

IntroductionChimeric antigen receptor (CAR) T cells show limited efficacy in solid tumors. Oncolytic viruses (OVs), especially those expressing immunomodulatory cytokines like interleukin-12 (IL-12), potentiate to synergize with CAR-T therapy.MethodsWe integrated an IL-12-expressing oncolytic herpes simplex virus type 2 (oHSV-2-IL-12) with mesothelin-targeting SS1-ICOSBBZ-CAR-T to treat Capan-2 pancreatic cancer cells xenografts in B-NDG immunodeficient mice.ResultsSS1-ICOSBBZ-CAR-T alone exhibited partial anti-tumor activity, but could not eradicate established tumors. Intra-tumoral oHSV-2-IL-12 administration potently enhanced CAR-T efficacy, achieving complete and durable tumor elimination even at reduced CAR-T doses. After the initial tumors were fully eliminated by combination therapy, mice were re-challenged by inoculating mesothelin-negative and mesothelin-positive tumor cell lines on the left and right flanks, respectively. In the combination treatment group, mesothelin-positive tumors failed to form new tumors within two weeks after re-challenge, whereas mesothelin-negative tumors grew normally. These findings indicate that oHSV-2-IL-12 combined with CAR-T therapy confers durable, antigen-specific protection against tumor re-challenge. Mechanistically, oHSV-2-IL-12 promoted CAR-T proliferation and persistence in peripheral blood and spleen. IL-12 expression also augmented the direct oncolytic effect of oHSV-2 in immunodeficient hosts.DiscussionThis synergistic approach achieves durable potent tumor clearance with reduced CAR-T doses, offering a transformative strategy against pancreatic cancer and other challenging solid malignancies.

Spinal cord injury (SCI) remains a significant clinical challenge and poses a dramatic threat to the life quality of patients due to limited neural regeneration and detrimental post-injury alternations in tissue microenvironment. We developed a therapeutic approach by transplanting spinal neural progenitor cells (spNPGs), derived from human induced pluripotent stem cell (iPSC)-generated neuromesodermal progenitors, into a contusive SCI model in NOD-SCID mice. Single-cell RNA sequencing mapped the in vitro differentiation of iPSC-spNPGs, confirming their specification into spinal neuronal lineages. Single-nucleus transcriptomics at 1 week post-transplantation showed that the grafted cells differentiated in vivo into motor neurons and two interneuron subtypes (V2 and dI4). Additionally, spNPGs integrated into host neural circuits, enhancing synaptic connectivity, while simultaneously modulating the injury microenvironment by shifting microglia and astrocyte polarization toward anti-inflammatory and neuroprotective phenotypes. This dual mechanism promoted axonal regrowth, remyelination, and significant sensorimotor recovery, as evidenced by improved locomotor scores. Our findings highlight the therapeutic potential of human iPSC-spNPGs in reconstructing neural networks and mitigating secondary damage, providing compelling preclinical evidence for advancing stem cell-based SCI therapies.

Human pluripotent stem cells (hPSCs) are a promising source for regenerative medicine due to their self-renewal and differentiation capacities. However, genetic instability acquired during reprogramming and in vitro culture presents major safety challenges for clinical translation. Recurrent mutations, especially structural variants (SVs), are of particular concern as they can impair differentiation and increase tumorigenic risk. In this review, we establish and systematically explore a central causal axis: SVs-three dimensional (3D) genome disruption-safety of hPSC-based therapy. We propose that SVs critically compromise therapeutic safety by perturbing the 3D architecture of the genome, leading to pathogenic rewiring of enhancer-promoter interactions. This rewiring, exemplified by "enhancer hijacking" and "enhancer loss," can aberrantly activate oncogenes or silence tumor suppressors even in the absence of copy number variations. Thus, 3D genome disruption provides a key mechanistic explanation for SV-driven tumorigenic potential and impaired differentiation fidelity in hPSCs. By highlighting this causal axis, our review not only advances the mechanistic understanding of SV-associated risks but also provides actionable insights for the development of more rigorous quality standards for hPSC-based cell therapy products.

Stem cell therapy is currently undergoing clinical research in China for conditions that are resistant to or incurable by traditional pharmaceutical interventions. Stem cell-derived extracellular vesicles (EVs) exhibit therapeutic effects similar to those of their parent stem cells, positioning them as an alternative or adjunctive approach to stem cell therapy. In recent years, given the tremendous potential for EVs in disease treatment, many researchers have focused on the development of stem cell-derived EVs and have achieved substantial progress in large-scale production and quality-related studies. However, at present, there are no specific or targeted regulatory requirements issued by authorities in China regarding the regulation of this novel therapeutic modality or the assurance of its safety and efficacy. In this paper, based on the biological properties of EVs, recent research advances, current understanding of their mechanisms of action, manufacturing processes and quality control strategies, a comprehensive framework for the quality evaluation of stem cell-derived EV-based therapeutic products is proposed. This framework is intended to serve as a reference for researchers and developers and may help to facilitate further discussion to facilitate further discussion, thereby supporting and promoting the development, regulatory oversight and establishment of quality standards and evaluation systems for stem cell-derived EVs in China.

Human mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) have demonstrated significant immunomodulatory and pro-regenerative potentials. However, the lack of specific markers to define MSC-sEVs presents a major challenge for their clinical application. Here, the proteomic datasets of MSC-sEVs from three cell sources were synchronously analyzed, and several surface antigens commonly found on MSCs were selected as candidate markers due to their high abundances in MSC-sEVs. Next, MSC-sEVs from three cell sources (adipose tissue, umbilical cord, and induced pluripotent stem cells) were stained with fluorescein-conjugated antibodies and analyzed by NanoFCM at single-vesicle resolution. The positive rates of CD13, CD29, and CD90 all exceeded 60% across sEVs derived from three MSCs sources, whereas other candidates generally exhibited lower positive rates. The high positive rates of them were further verified in MSC-sEVs purified via other methods. Moreover, high-resolution microscopy, as an orthogonal method, visually validated their high presence in MSC-sEVs. Meanwhile, none of the non-MSC-sEVs showed concurrent positive rates for CD13, CD29, and CD90 exceeding 40%, suggesting that this marker panel (with a positive rate threshold of 50% for all three markers) could specifically distinguish MSC-sEVs from non-MSC-sEVs. Finally, the positive rates of this panel of markers were assessed in sEVs derived from MSCs at successive passages. The results revealed a progressive diminution of the positive rates of CD29 and CD90 in the sEVs secreted by MSCs with successive passages, accompanied by a reduction in the pro-proliferative activity of these sEVs. Taken together, we have identified a specific and quantifiable marker panel for MSC-sEVs characterization, which facilitates the development of standardized assays for defining MSC-sEVs and ultimately accelerates the clinical translation of MSC-sEVs.

Experimental autoimmune encephalomyelitis (EAE) serves as a model for studying multiple sclerosis, with immunization strategies utilizing myelin oligodendrocyte glycoprotein (MOG)35-55 peptide, emulsified in adjuvant enriched with Mycobacterium tuberculosis (Mtb). This study examined the effects of Bacillus Calmette-Gu & eacute;rin (BCG) as an adjuvant, alongside the impact of MOG 35-55 peptide doses and their residual counter ions on EAE development. We found that BCG can be effectively used to induce EAE with similar incidence and severity as heat-killed H37Ra, contingent upon the appropriate MOG 35-55 peptide dose. Different immunization doses of MOG 35-55 peptide significantly affect EAE development, with higher doses leading to a paradoxical reduction in disease activity, probably due to peripheral tolerance mechanisms. Furthermore, doses of MOG 35-55 peptides with acetate showed a more pronounced effect on disease development compared to those containing trifluoroacetic acid (TFA), suggesting the potential influence of residual counter ions on EAE activity. We highlighted the feasibility of applying BCG to the establishment of EAE for the first time. Our findings emphasized the importance of MOG 35-55 peptide dosage and composition in modulating EAE development, offering insights into the mechanisms of autoimmunity and tolerance. This could have implications for autoimmune disease research and the design of therapeutic strategies.

Cell therapy products (CTPs) have become a highly promising area within the realm of regenerative medicine research in China. In recent years, there has been a notable increase in the demand for new drug applications related to these innovative products. However, the diverse range of sources, biological functions, product types, and technologies associated with CTPs present considerable regulatory challenges. This article delves into how regulatory authorities in China have been continuously refining their frameworks, enhancing quality evaluation abilities, and fostering open communication and collaboration with the industry to tackle these challenges. These ongoing regulatory efforts have played a crucial role in facilitating the healthy and rapid growth of the clinical translation of CTPs in China.

The generation of an immune response in neoantigen-based products relies on antigen presentation, which is closely analyzed by bioassays for T-cell functions such as tetramer or cytokine release. Mass spectrometry (MS) has the potential to directly assess the antigen-presenting capability of antigen-presenting cells (APCs), offering advantages such as speed, multi-target analysis, robustness, and ease of transferability. However, it has not been used for quality control of these products due to challenges in sensitivity, including the number of cells and peptide diversity. In this study, we describe the development and validation of an improved targeted LC-MS/MS method with high sensitivity for characterizing antigen presentation, which could be applied in the quality control of neoantigen-based products. The parameters for the extraction were carefully optimized by different short peptides. Highly sensitive targeted triple quadrupole mass spectrometry combined with ultra-high performance liquid chromatography (UHPLC) was employed using a selective ion monitoring mode (Multiple Reaction Monitoring, MRM). Besides, we successfully implemented robust quality control peptides to ensure the reliability and consistency of this method, which proved invaluable for different APCs. With reference to the guidelines from ICH Q2 (R2), M10, as well as considering the specific attributes of the product itself, we validated the method for selectivity, specificity, sensitivity, limit of detection (LOD), recovery rate, matrix effect, repeatability, and application in dendritic cells (DCs) associated with neoantigen-based products. The validation process yields satisfactory results. Combining this approach with T cell assays will comprehensively assess cell product quality attributes from physicochemical and biological perspectives.

Experimental autoimmune encephalomyelitis (EAE) serves as a model for studying multiple sclerosis, with immunization strategies utilizing MOG

Lentiviruses, with their high transduction efficiency and gene expression levels, are widely used as gene delivery vectors in the development of chimeric antigen receptor T cells (CAR-T) and other genetically modified cell therapies. Accurate determination of the lentiviral vector infectious titer is essential to ensure effective transduction and product consistency. In this study, we developed an efficient method for lentiviral vector titration based on digital droplet polymerase chain reaction (ddPCR) technology, enabling absolute quantification of the target gene. Benzonase treatment of non-transduced plasmids substantially shortened the experimental period, reducing cell culture duration from 10-14 days-3 days. The method was rigorously validated by assessing specificity, working range, limit of quantification, precision, accuracy, and robustness. This study demonstrates the feasibility of combining enzymatic digestion with ddPCR to quantify lentiviral vector infectious titer and provides a detailed and readily adaptable methodology for the scientific community.

Objective:To evaluate the feasibility of 8E5 cells and CD19-CAR-Jurkat cells used as reference cells in the detection of lentiviral vector integration sites with different methods.Methods:Single clones of 8E5 cells and CD19-CAR-Jurkat cells were selected using limiting dilution method. Digital PCR was established to detect the copy number of HIV-1 in 8E5 cells and the copy number of CAR in CD19-CAR-Jurkat cells. High-throughput sequencing techniques (whole-genome resequencing, modified genome sequencing and probe hybridization capture) were used to detect integration sites in 8E5 cells and CD19-CAR-Jurkat cells, and optical genome mapping (OGM) technology was used for further confirmation.Results:Three clones of 8E5-D8 cells and six clones of CD19-CAR-Jurkat 2-6 cells were selected using the limiting dilution method. 8E5-D8 and CD19-CAR-Jurkat 2-6 were chosen as candidate cells based on their gene copy numbers detected by digital PCR and flow cytometry. These cells were then expanded and cryopreserved. Digital PCR showed that 8E5-D8 cells contained approximately 1 copy per cell, while CD19-CAR-Jurkat 2-6 cells contained approximately 13 copies per cell. High-throughput sequencing revealed one integration site in 8E5 cells and 13 integration sites in CD19-CAR-Jurkat cells, which matched the copy number detection results. All these integration sites were further confirmed at the submicroscopic level of chromosomes using OGM.Conclusions:Based on the insertion copy numbers and integration sites, 8E5-D8 cells and CD19-CAR-Jurkat 2-6 cells could be used as reference cells in further development of methods for detecting integration sites in CAR-T cell lentiviral vectors.

Efficient and large-scale expansion of mesenchymal stem/stromal cells (MSCs) has always been a formidable challenge to researchers in cell-based therapies and regenerative medicine. To reconcile major drawbacks of 2D planar culturing system, we innovatively developed an automated closed industrial scale cell production (ACISCP) platform based on GMP-grade microcarrier for culture of umbilical cord-mesenchymal stem/stromal cells (UCMSCs), in accordance with the criteria of stem cell bank. ACISCP system is a fully closed system, which employs different models of vivaSPIN bioreactors (CytoNiche Biotech, China) for scale-up cell culture and vivaPREP (CytoNiche Biotech, China) for automated cell harvesting and cell dosage preparation. To realize industrial scale expansion of UCMSCs, a three-stage expansion was conducted with 1 L, 5 and 15 L vivaSPIN bioreactors. Using 3D TableTrix® and ACISCP system, we inoculated 1.5 × 107 of UCMSCs into 1 L vivaSPIN bioreactor and finally scaled to two 15 L bioreactor. A final yield of 2.09 × 1010 cells with an overall expansion factor of 1975 within 13 days. The cells were harvested, concentrated, washed and prepared automatically with vivaPREP. The entire process was realized with ACISCP platform and was totally enclosed. Critical quality attributes (CQA) assessments and release tests of MSCs, including sterility, safety, purity, viability, identity, stability and potency were performed accordingly. The quality of cells harvested from 3D culture on the ACISCP and conventional 2D planar culture counterpart has no significant difference. This study provides a bioprocess engineering platform, harnessing GMP-grade 3D TableTrix® microcarriers and ACISCP to achieve industrial-scale manufacturing of clinical-grade hMSCs.

本文阐述了《中国药典》2020年版新增通则"生物制品病毒安全性控制"的原则和框架起草考量以及该通则的作用、地位和意义.

Objective:To establish VSV-G qPCR assay for detection of replication competent lentivirus(RCL) and verify its application.Methods:A real-time fluorescent quantitative PCR for VSV-G envelope gene was developed. Several parameters including specificity, linear, amplification efficiency, precision, trueness, dynamic range, limit of detection, limit of quantification and robustness were verified. Preliminary application on CAR-T cells, end of production cells and the harvest of lentivirus vector was performed by using the method developed.Results:The real-time fluorescent quantitative PCR assay for VSV-G was specific for the detection VSV-G without specific amplification on 293T, PBMC and C8166 cells. The linear range of the assay was 1×10 2 copies/test-1×10 9 copies/test with a R2 value more than 0.998 and amplification efficiency between 93% and 98%. The precision (relative standard deviation) of the assay was less than 12% and the trueness (the rate of recovery) of the assay was between 85% and 106%. The limit of detection (LOD) and limit of quantification (LOQ) of the assay was 5 copies/test and 40 copies/test. In addition, the robustness of the assay was also well. All the results of validation illustrated that the assay could meet the detection requirements. All of the 54 samples including CAR-T cells, lentivirus vector and end of product cells after amplification and passage on C8166 cells were negative of RCL by using the established assay. Conclusions:The real-time fluorescent quantitative PCR for VSV-G were established successfully. All of the validation results illustrated that the assay could meet the detection requirements. The application of the assay was conducive to further enhance the safety of the lentivirus vector related products.

目的:建立可定量检测质粒DNA超螺旋构象相对百分含量的毛细管凝胶电泳法(CGE),并开展方法学验证研究及初步应用探讨.方法:选用涂层毛细管,采用荧光检测器CGE法分离质粒DNA,首先通过限制性内切酶和缺刻酶处理法确认质粒DNA超螺旋(covalently closed circular,ccc)、线性(linear)及开环(open circular,oc)这3种构象在电泳图谱中的位置,然后通过对荧光染料、样品上样浓度等条件进行优化,建立质粒DNA构象的检测方法.对所建方法进行专属性、准确性、线性、精密度、检测下限和定量下限验证,并通过将质粒DNA置于不同保存温度、反复冻融及紫外照射后再进行质粒DNA构象的检测,初步分析该方法应用的可行性.结果:采用总长度为40 cm、有效长度为30.2 cm涂层毛细管,毛细管和样品上样温度分别为20℃和4℃,质粒浓度为4 ng·μL-1以1 378.95 Pa,4 s上样分离,CGE法可准确地定量质粒DNA 3种构象所占的比例,且分离度良好.通过对3种常用的质粒保存液基质的检测结果显示,这些基质不会干扰检测结果,专属性较好.质粒DNA3种构象的线性范围均在0.25～8 ng·μL-1,构象峰面积与上样浓度具有良好的线性关系,相关系数分别是0.997 7、0.999 4、0.992 7.将线性化质粒及开环质粒分别以3％～50％添加至原质粒中,可检出的线性及开环构象的峰面积与添加浓度呈现良好的线性关系,相关系数分别为0.9999和0.997 2,线性构象的回收率在104％～120％,准确性良好;开环构象的回收率稍差,为68％～94％.将质粒DNA分别以高(4μg·mL-1)、中(2μg·mL-1)、低(0.5 μg·mL-1)3个浓度验证实验的重复性,结果显示,试验内重复性良好,质粒DNA超螺旋构象迁移时间的RSD在0.22％～0.54％,相对百分含量的RSD在0.38％～2.1％;中间精密度也较好,迁移时间的RSD在0.52％～1.1％;相对百分含量的精密度在质量浓度为2 ng·μL-1和4 ng·μL-1时,RSD值分别为3.6％、0.25％.灵敏度的验证结果显示,定量下限及检测下限分别为0.5 ng·μL-1和0.25 ng·μL-1.用该方法分别检测质粒DNA在不同保存条件、冻融及紫外照射后的构象变化,结果显示,质粒DNA在-80℃、-20℃保存1周,超螺旋构象无明显变化,但在室温保存1周后,质粒DNA开环构象含量从1.39％增加到4.16％;将质粒DNA反复冻融5次,未观察到明显的构象变化,但冻融7次后,质粒DNA开环构象相对百分含量从1.3％左右增加到1.93％;质粒DNA质量浓度在1 mg·mL-1时,紫外照射15 min到3 h,质粒DNA的超螺旋构象相对百分含量从96.97％下降到54.38％,同时线性及开环构象明显增加.结论:本研究建立的毛细管凝胶电泳法可以很好地将质粒DNA的超螺旋、线性及oc 3种构象分离,各构象分离度良好,可准确定量不同构象相对百分含量,线性相关系数不低于0.99,灵敏度高,可重复性好,且可灵敏地反映出质粒DNA构象的变化,可用于质粒DNA构象纯度检测的质量控制.

The outbreak of COVID-19 has posed a huge threat to global health and economy. Countermeasures have revolutionized norms for working, socializing, learning, and travel. Importantly, vaccines have been considered as most effective tools to combat with COVID-19. As of the beginning of 2021, >200 COVID-19 vaccine candidates, covering nearly all existing technologies and platforms, are being research and development (R&D) by multiple manufacturers worldwide. This has posed a huge obstacle to the quality control and evaluation of those candidate vaccines, especially in China, where five vaccine platforms are deployed in parallel. To accelerate the R&D progress of COVID-19 vaccines, the guidances on R&D of COVID-19 vaccine have been issued by National Regulatory Authorities or organizations worldwide. The Center for Drug Evaluation and national quality control laboratory in China have played a leading role in launching the research on quality control and evaluation in collaboration with relevant laboratories involved in the vaccine R&D, which greatly supported the progression of vaccines R&D, and accelerated the approval for emergency use and conditional marketing of currently vaccine candidates. In this paper, the progress and experience gained in quality control and evaluation of COVID-19 vaccines developed in China are summarized, which might provide references for the R&D of current and next generation of COVID-19 vaccines worldwide.

Mesenchymal stromal cell (MSC)-based therapies are being actively investigated in various inflammatory disorders. However, functional variability among MSCs cultured in vitro will lead to distinct therapeutic efficacies. Until now, the mechanisms behind immunomodulatory functional variability in MSCs are still unclear. We systemically investigated transcriptomic variations among MSC samples derived from multiple tissues to reveal their effects on immunomodulatory functions of MSCs. We then analyzed transcriptomic changes of MSCs licensed with INFγ to identify potential molecular mechanisms that result in distinct MSC samples with different immunomodulatory potency. MSCs were clustered into distinct groups showing different functional enrichment according to transcriptomic patterns. Differential expression analysis indicated that different groups of MSCs deploy common regulation networks in response to inflammatory stimulation, while expression variation of genes in the networks could lead to different immunosuppressive capability. These different responsive genes also showed high expression variability among unlicensed MSC samples. Finally, a gene panel was derived from these different responsive genes and was able to regroup unlicensed MSCs with different immunosuppressive potencies. This study revealed genes with expression variation that contribute to immunomodulatory functional variability of MSCs and provided us a strategy to identify candidate markers for functional variability assessment of MSCs.

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 mu g or 50 mu g) or placebo intramuscularly, 30 days apart. In phase 2, participants were randomly assigned (1:1:1:1:1:1) to receive the vaccine (25 mu g or 50 mu g) 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 mu g vaccine (n=20), or the 50 mu g 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 mu g vaccine (n=150), or 50 mu g vaccine (n=150), or three doses of placebo (n=150), 25 mu g vaccine (n=150), or 50 mu g 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 mu g group, and 18 [90%] of 20 in the 50 mu g group; phase 2: 37 [25%] of 150 in the two-dose placebo group, 43 [29%] of 150 in the two-dose 25 mu g group, 50 [33%] of 150 in the two-dose 50 mu g group, 47 [31%] of 150 in the three-dose placebo group, 72 [48%] of 150 in the three-dose 25 mu g group, and 65 [43%] of 150 in the three-dose 50 mu g group). In phase 1, two (10%) grade 3 or worse adverse events were reported in the 50 mu g group. In phase 2, grade 3 or worse adverse events were reported by 18 participants (four [3%] in the two-dose 25 mu g vaccine group, two [1%] in the two-dose 50 mu g vaccine group, two [1%] in the three-dose placebo group, four [3%] in the three-dose 25 mu g vaccine group, and six [4%] in the three-dose 50 mu g vaccine group), and 11 were considered vaccine related (two [1%] in the two-dose 25 mu g vaccine group, one [1%] in the two-dose 50 mu g vaccine group, one [1%] in the three-dose placebo group, two [1%] in the three-dose 25 mu g vaccine group, and five [3%] in the three-dose 50 mu g vaccine group); seven participants reported serious adverse events (one [1%] in the two-dose 25 mu g vaccine group, one [1%] in the two-dose 50 mu g vaccine group, two [1%] in the three-dose placebo group, one [1%] in the three-dose 25 mu g vaccine group, and two [1%] in the three-dose 50 mu g 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 mu g group and 72% (108 of 150) in the 50 mu g 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 mu g group and 93% (138 of 148) in the 50 mu g 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 mu g group and 14.1 (10.8-18.3) in the 50 mu g 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 mu g group and 69.1 (53.0-90.0) in the 50 mu g 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 mu g dose in a three-dose schedule in an ongoing phase 3 trial for large-scale evaluation of ZF2001's safety and efficacy. Copyright (C) 2021 Elsevier Ltd. All rights reserved.