Background Mucosal immunity constitutes the primary defense against pathogens. Vaccination strategies aimed at inducing mucosal immunity are therefore crucial for preventing viral infections. Lipid nanoparticle (LNP) delivery systems enhance mRNA vaccine stability, giving them the potential for mucosal delivery.Methods This study employed an mRNA-LNP vaccine candidate encoding the SARS-CoV-2 Spike protein, compatible with intranasal delivery, to evaluate immunization strategies aimed at inducing robust systemic and mucosal immunity. Antibody and T cell responses in both systemic and mucosal compartments, as well as protection against viral challenge, were assessed in mice following various regimens, including single-route and heterologous prime-boost strategies. Single B-cell immune repertoire analysis was performed to further elucidate the advantages of an optimized mucosal immunization approach.Results Intranasal immunization alone induced weak systemic antibody responses and failed to elicit mucosal immunity. In contrast, an "intramuscular prime and intranasal boost" regimen provoked strong systemic and mucosal immunity, marked by significantly elevated levels of antigen-specific T cells and mucosal IgA, which correlated with broader protection. Immunoprofiling revealed that intranasal boosting promoted IgA class switching in antigen-specific B cells and diversified the antigen-specific B cell receptor (BCR) repertoire.Conclusion This study demonstrates that mRNA vaccines encapsulated in tailored LNPs can be effectively delivered via an "intramuscular prime and intranasal boost" strategy, establishing an efficacious approach for mRNA mucosal immunization.

The Epstein-Barr virus (EBV) is associated with a range of diseases, including malignancies and autoimmune disorders. Driven by advances in deep sequencing technologies, recent studies have systematically characterized EBV genomic landscapes in diverse clinical specimens, uncovering distinct geographic patterns in strain distribution. However, a comprehensive understanding of how EBV genomic variation contributes to disease pathogenesis remains incomplete. This review aims to consolidate current knowledge on EBV genomic variation and its role in disease development. First, we delineate the evolutionary origins of EBV genetic variations, focusing on key factors such as replication errors, recombination, and immune-driven selection. Next, we summarize the association between EBV subtypes and diseases, with evidence linking specific genetic variations to malignancies, infectious mononucleosis, and multiple sclerosis. At the mechanistic level, EBV genomic variation may influence pathogenesis through three interconnected mechanisms: viral functional changes, viral-host interactions, and molecular mimicry. Finally, this review explores how genomic insights into EBV can inform clinical applications, including early diagnostic biomarkers and next-generation therapies targeting lineage-specific variations, such as mRNA vaccines and small molecules that disrupt latency. These advances highlight the critical role of EBV genomics in understanding disease mechanisms and developing precision interventions to address the global burden of EBV-related diseases.

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.

Introduction:Vaccines capable of effectively inducing mucosal immunity, particularly specific IgA antibodies, represent an ideal strategy for preventing infections and the transmission of pathogens such as SARS-CoV-2 and influenza viruses that rapidly replicate in the upper respiratory tract and cause clinical symptoms. However, a lack of standardized nasal antibody detection and sampling methods has hindered cross-study comparability and vaccine development. Methods:This study uses SARS-CoV-2 as a model pathogen to standardize nasal antibody detection methods and sampling methods. Following the scientific guidelines (Q14 and Q2(R2)) for analytical procedure development and validation released by the International Council for Harmonization (ICH), an ELISA for nasal SARS-CoV-2 WT-RBD specific IgA detection was established and validated. To compare the sampling methods, nasal samples were collected from five groups using three commonly used nasal sampling methods (M1: nasopharyngeal swab; M2: nasal swab; M3: expanding sponge method). The total IgA and SARS-CoV-2 WT-RBD IgA in clinical samples were detected. Results:The first validated ELISA for nasal SARS-CoV-2 WT-RBD specific IgA detection was established through analytical target profiling (ATP), risk assessment, and design of experiment optimization. Systematic validation demonstrated exclusive specificity for the target antigen, with intermediate precision of <17% and relative bias of <±4%, meeting ATP requirements. Analysis of 154 clinical samples demonstrated strong concordance between the novel method and electrochemiluminescence assays, with a concordance correlation coefficient of 0.87 for quantitative results and a kappa coefficient of 0.85 for results above and below the dilution-adjusted limit of quantification (LOQ). Applying this novel method, a clinical comparison revealed that M3 achieved superior performance in terms of the single-day detection rate (above dilution-adjusted LOQ 95.5%), 5-day consecutive detection rate (above dilution-adjusted LOQ 88.9%), and median SARS-CoV-2 WT-RBD IgA concentration (171.2 U/mL), significantly outperforming M1 (68.8%; 48.7%; 28.7 U/mL, p<0.0001) and M2 (88.3%; 77.3%; 93.7 U/mL, p<0.05). Conclusion:This study has established the first standardized nasal detection system. The system can be adapted with appropriate modifications for the clinical evaluation of other respiratory mucosal vaccines, thereby advancing the development of mucosal vaccines.

INTRODUCTION:Double-stranded RNA (dsRNA) is a key impurity of mRNA vaccines prepared by in vitro transcription (IVT) and is primarily transcribed by T7 RNA polymerase. It can trigger innate immunity and induce a series of side effects that may influence the safety of mRNA vaccines. AREAS COVERED:This manuscript summarizes dsRNA generation mechanisms and immunity activation and analyzes the current challenges in dsRNA detection and control strategies. Regulatory standards for dsRNA impurities in mRNA vaccines have also been discussed. EXPERT OPINION:dsRNA as a critical quality attribute (CQA), the structural heterogeneity of it (including length and structure) and its precise immunomodulatory mechanisms affecting vaccine safety are poorly understood. Regulatory authorities have not released specific standards for dsRNA. Additionally, there is a lack of comparative analysis data on different corporate testing methods. Therefore, to ensure the safety of dsRNA containing mRNA vaccines and improve mRNA-based platforms, it is of great significance to establish standardized detection methods and standards for dsRNA; to design mRNA production with low dsRNA impurities by adopting the quality by design (QbD) approach; and to evaluate the immune stimulation mechanism of dsRNA impurities in mRNA vaccines.

Since 2019, there has been a growing focus on mRNA vaccines for infectious disease prevention, particularly following the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). mRNA vaccines offer advantages such as rapid production and the ability to induce robust cellular and antibody responses, which are essential for combating infections that require cell-mediated immunity, including Tuberculosis (TB). This review explores recent progress in TB mRNA vaccines and addresses several key areas: (1) the urgent need for new TB vaccines; (2) current advancements in TB vaccine development, and the advantages and challenges of mRNA technology; (3) the design and characteristics of TB mRNA vaccines; (4) the immunological mechanisms of TB mRNA vaccines; (5) manufacturing processes for TB mRNA vaccines; and (6) safety and regulatory considerations. This interdisciplinary review aims to provide insights for researchers working to address critical questions in TB mRNA vaccine development.

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.

Messenger RNA (mRNA) vaccines have revolutionized infectious disease prevention and cancer immunotherapy due to their rapid development, potent immunogenicity, and flexible design. Central to the clinical success of mRNA vaccines, lipid nanoparticles (LNPs) function as efficient, nonviral delivery systems capable of protecting mRNA and facilitating its uptake by target cells. Recent advances have demonstrated that LNP-formulated mRNA vaccines and therapeutics elicit robust immune responses and confer effective protection against a broad spectrum of pathogens, including viruses and bacteria. Moreover, LNP-based therapies have shown promising therapeutic efficacy in various cancers and rare diseases, as evidenced by both preclinical models and clinical trials. This review provides a comprehensive overview of the key components, structural features, and preparation technologies of LNPs. It further discusses ongoing challenges in LNP design, such as delivery efficiency, tissue targeting, and safety, and proposes rational strategies to address these limitations. Additionally, recent progress in the analytical methods used to characterize the critical quality attributes of LNPs is highlighted. This review aims to guide the rational design of next-generation LNPs and to support the broader application of mRNA-based vaccines and therapeutics.

Mucosal immunity is crucial for preventing the infection and transmission of respiratory viruses. Nasal antibody is inversely correlated with a lower risk of infection with respiratory viruses. However, the current reference standard for nasal antibody assessment is serum-based, mainly consisting of monomeric IgG and IgA. The applicability of serum-derived standards for assessing nasal antibodies, consisting mostly of dimeric or polymeric secretory IgA (sIgA), remains unvalidated. Herein, we first proved that the sera-derived standard was not applicable for assessing nasal antibodies. Using a non-homologous standard as a calibrator introduced systematic error up to 10 times, which did not benefit the understanding of mucosal antibody response. Therefore, we attempted to develop two candidate standards (CS1, CS2) using nasal mucosal lining fluids (NMLFs) collected from SARS-CoV-2 Omicron convalescents or intranasal vaccine recipients, and CS3 using a sIgA monoclonal antibody. CS2 exhibited broad-spectrum binding activity against 12 SARS-CoV-2 strains, including all tested Omicron subvariants. A collaborative study conducted by seven laboratories demonstrated that CS2 improved the harmonization of inter-laboratory variability (pre-standardization geometric coefficients of variance, 14-314%; post-standardization, 3-35%). Using CS2 ensured an accurate assessment of nasal antibodies. Thus, CS2 was established as a national standard for evaluating nasal SARS-CoV-2-specific antibodies (Lot: 300052-202401, 1000 U/mL). Our work provides a benchmark for evaluating mucosal vaccines for SARS-CoV-2 and inspires new avenues for developing new reference standards for other mucosal vaccines.

OBJECTIVE:This study aimed to evaluate the vaccination status of the National Immunization Program (NIP) among children born in 2020-2021 in China and assess the impact of the COVID-19 pandemic on vaccination coverage. METHODS:Three counties were randomly selected from each of the provinces of Zhejiang, Chongqing, and Shanxi in China. Vaccination records were retrieved from provincial immunization information systems. Statistical analyses were conducted using descriptive epidemiological methods and chi-square tests to compare vaccination coverage across different regions and birth cohorts. The Kaplan-Meier method was also applied to estimate vaccination coverage at specific ages. RESULTS:Except DTaP4, NIP vaccination coverage for children born in 2020-2021 remained high, exceeding the 90.00 % target. Coverage ranged from 90.09 % for PV3 (95 % CI, 90.07-90.47 %) to 99.21 % for HepB1 (95 % CI, 99.15-99.27 %). However, compared to children born during 2015-2019, coverage for most vaccines (excluding BCG and HepB1) among those born in 2020-2021 significantly declined (all P-values <0.05). Regarding timeliness, the timely vaccination coverage for NIP vaccines ranged from 78.96 % (95 % CI, 78.70-79.25 %) for DTaP4 to 97.74 % (95 % CI, 97.65-97.84 %) for HepB2. The primary reasons for vaccination delays were missed doses (54.9 %) and delayed administration (41.0 %). Significant regional differences in vaccination coverage and timeliness were also observed (all P-values <0.05). CONCLUSIONS:Despite pandemic challenges, China maintained high NIP coverage, though some declines occurred. Regional differences highlight the need for targeted interventions to improve immunization services, ensure timely protection, and address socioeconomic and health resource disparities.

Tuberculosis (TB) remains one of the most significant challenges to global public health. Vaccine development is a critical strategy for the prevention and control of TB. However, evaluating the protective efficacy of TB vaccines faces numerous challenges, particularly in the selection of animal models and bacterial strains. Variations in animal models, challenge strains, challenge routes, and doses can significantly impact the outcomes of preclinical evaluations. This article highlights the importance of standardizing preclinical evaluation models, summarizes the animal models and challenge strains used in novel TB vaccine candidates, efficacy studies, and discusses the advantages and limitations of commonly used animal models in TB vaccine research. It also points out the differential performance of various animal models in simulating protection and pathology. Given the current limitations of using a narrow range of challenge strains and the lack of standardized infection routes and doses, this article calls for the establishment of more standardized challenge strains and the development of standardized evaluation models to improve the reliability and generalizability of new TB vaccine efficacy assessments.

Background: Self-amplifying mRNA (saRNA) holds promising application prospects. However, due to the inclusion of a replicase sequence, its extended length leads to premature termination during in vitro transcription (IVT), resulting in poor product integrity. This study aims to optimize the IVT process for saRNA vaccines to enhance integrity, thereby addressing the key challenge in saRNA vaccine manufacturing. Method: Guided by the Quality by Design (QbD) framework, Design of Experiment (DoE) methodology was employed to design diverse combinations of process parameters for IVT reactions. Predictive models were established to identify critical process parameters (CPPs) influencing integrity and yield. An optimized parameter set and process design space, meeting predefined yield and integrity standards, were developed. The impact of integrity on the immunogenicity of saRNA vaccines was further investigated. Results: Mg2+ concentration exerted the most pronounced effect on saRNA integrity. Under optimized IVT conditions, integrity exceeded 85%. Mathematical modeling simulations defined the IVT design space, meeting the preset criteria of ≥80% integrity and ≥600 μg/100 μL yield while accommodating longer saRNA constructs. Notably, murine model data revealed that higher saRNA integrity significantly enhanced antigen-specific antibody and T-cell responses. Conclusion: This study successfully established a multivariate IVT design space fulfilling preset integrity and yield criteria, providing critical data references for the industrialization and quality specification development of saRNA vaccines.

To identify nasopharyngeal carcinoma (NPC)-relevant T cell receptors (TCRs), we profile the repertoires of peripheral blood TCRβ chains from 228 NPC patients, 241 at-risk controls positive for serum Epstein-Barr virus (EBV) VCA-IgA antibody, and 251 seronegative controls. We develop a TCR-based signature (T-score) based on 208 NPC-enriched CDR3β sequences, which accurately diagnoses NPC in both the original and independent validation cohorts. Notably, a higher T-score, associated with a shorter time interval to NPC diagnosis, effectively identifies early-stage NPC among EBV-seropositive at-risk individuals prior to clinical diagnosis. These NPC-enriched TCRs react against not only EBV-specific antigens but also non-EBV antigens expressed by NPC cells, indicating a broad range of specificities. Moreover, the abundance of NPC-enriched CD8+ T cells in blood correlates with the infiltration of non-exhausted T cell counterparts in tumors and predicts prolonged survival, suggesting that these NPC-enriched T cells have significant potential for disease monitoring and therapeutic applications.

Objective There are limited data on the use of the creation tuberculin skin test (C-TST) for detecting tuberculosis (TB) infection (TBI) in individuals under 18 years of age. We conducted a study to assess the diagnostic accuracy of C-TST in this population. Methods A double-blind, randomized controlled trial was conducted across 4 tertiary hospitals in China to evaluate the diagnostic accuracy of the C-TST in detecting TBI in individuals under 18 years of age. Participants with suspected pulmonary TB, extrapulmonary TB, or non-TB pulmonary disease were enrolled. The primary outcome was the diagnostic accuracy of the C-TST. Secondary outcomes included the consistency among C-TST, the traditional tuberculin skin test (TST), and T-SPOT.TB assays in different subgroups, as well as the safety of C-TST. Each participant underwent all 3 tests simultaneously: T-SPOT.TB assay, TST, and C-TST. Results C-TST showed a sensitivity of 83.0 % (95 % CI, 68.7 %–91.9 %), while TST and T-SPOT.TB demonstrated sensitivities of 80.9 % (95 % CI, 66.3 %–90.4 %) and 76.6 % (95 % CI, 61.6 %–87.2 %), respectively. The specificities of C-TST, TST, and T-SPOT.TB were 100 % (95 % CI, 91.9 %–100 %), 98.0 % (95 % CI, 87.8 %–99.9 %), and 100 % (95 % CI, 90.9 %–100 %), respectively. The consistency between C-TST and T-SPOT.TB was high (kappa = 0.847). No serious adverse events (AEs) were reported. Conclusions This study demonstrates that C-TST is a reliable and safe diagnostic tool for detecting TBI in children and adolescents. It shows higher sensitivity than both T-SPOT.TB and the traditional TST, with no associated serious AEs. Therefore, C-TST is an effective and safe option for diagnosing TBI in this age group.

The experience of fighting COVID-19 shows the growing importance of vaccine-induced cellular immunity, whereas aluminum adjuvants fail to meet this medical requirement. Herein, with very simple processing, we prepared a narrow lateral size of monolayer clay nanosheets from montmorillonite, a FDA-approved generally recognized as safe (GARS) material. The nanosheets adsorbed antigen ovalbumin (OVA) through hydrophilic and electrostatic interactions, while utilizing nanosheets to expose more octahedral Al-OH and tetrahedral Si-OH residues to complete the adsorption of mannose (Man) targeting antigen presentation cells, leading to the production of nanosheet vaccines. In additional, the lyophilization process did not significantly affect the loading of OVA and Man on the nanosheets. The obtained nanosheet vaccines induced a strong cellular immune response in addition to a potent humoral immune response. Inspired by the results, the prophylactic nanosheet adjuvant vaccine against Epstein-Barr virus (EBV) was developed, which induced a 13-fold increase in EBV-neutralizing antibody titers compared to aluminum adjuvant vaccine, and efficiently blocked EBV infection of target cells. Based on the simple formulation process, remarkable immune response effect and excellent safety profile, montmorillonite nanosheets have the potential to replace aluminum adjuvant as a vaccine platform for inducing effective immune responses.

IntroductionFollowing the coronavirus disease pandemic, respiratory mucosal vaccines that elicit both mucosal and systemic immune responses have garnered increasing attention. However, human physiological characteristics pose significant challenges in the evaluation of mucosal immunity, which directly impedes the development and application of respiratory mucosal vaccines.Areas CoveredThis study summarizes the characteristics of immune responses in the respiratory mucosa and reviews the current status and challenges in evaluating immune response to respiratory mucosal vaccines.Expert OpinionSecretory Immunoglobulin A (S-IgA) is a major effector molecule at mucosal sites and a commonly used indicator for evaluating respiratory mucosal vaccines. However, the unique physiological structure of the respiratory tract pose significant challenges for the clinical collection and detection of S-IgA. Therefore, it is imperative to develop a sampling method with high collection efficiency and acceptance, a sensitive detection method, reference materials for mucosal antibodies, and to establish a threshold for S-IgA that correlates with clinical protection. Sample collection is even more challenging when evaluating mucosal cell immunity. Therefore, a mucosal cell sampling method with high operability and high tolerance should be established. Targets of the circulatory system capable of reflecting mucosal cellular immunity should also be explored.

Neutralizing antibodies (NtAbs) against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are indicators of vaccine efficacy that enable immunity surveillance. However, the rapid mutation of SARS-CoV-2 variants prevents the timely establishment of standards required for effective XBB vaccine evaluation. Therefore, we prepared four candidate standards (No. 11, No. 44, No. 22, and No. 33) using plasma, purified immunoglobulin, and a broad-spectrum neutralizing monoclonal antibody. Collaborative calibration was conducted across nine Chinese laboratories using neutralization methods against 11 strains containing the XBB and BA.2.86 sublineages. This study demonstrated the reduced neutralization potency of the first International Standard antibodies to SARS-CoV-2 variants of concern against XBB variants. No. 44 displayed broad-spectrum neutralizing activity against XBB sublineages, effectively reduced interlaboratory variability for nearly all XBB variants, and effectively minimized the geometric mean titer (GMT) difference between the live and pseudotyped virus. No. 22 showed a broader spectrum and higher neutralizing activity against all strains but failed to reduce interlaboratory variability. Thus, No. 44 was approved as a National Standard for NtAbs against XBB variants, providing a unified NtAb measurement standard for XBB variants for the first time. Moreover, No. 22 was approved as a national reference reagent for NtAbs against SARS-CoV-2, offering a broad-spectrum activity reference for current and potentially emerging variants.

This study aimed to evaluate the diagnostic performance of ESAT6-CFP10 (EC) skin test in healthy population and determine the factors influencing the booster effect. We conducted a randomized, double-blind, parallel controlled trial in healthy population. The experiment was divided into two stages. In the first stage, all participants underwent T-SPOT, TB-PPD, and EC tests. In the second stage, to evaluate whether BCG vaccination affected the efficacy of skin tests, the participants with three negative results in the first stage were randomly assigned to the BCG and placebo groups at a ratio of 2:1 and underwent three tests. The positivity rates and concordance of the three tests were calculated in both stages, and a logistic regression model was constructed to determine the factors influencing the booster effect of EC in the second stage. Safety observations were continued until the skin test results were available. In the first stage, 1,564 participants were enrolled in the study. The positivity rates of the T-SPOT, EC, and TB-PPD tests in all the participants were 18.89

Circular RNA (circRNA) vaccines have attracted increasing attention due to their stable closed-loop structures and persistent protein expression ability. During the synthesis process, nicked circRNAs with similar molecular weights to those of circRNAs are generated. Analytical techniques based on differences in molecular weight, such as capillary electrophoresis, struggle to distinguish between circRNAs and nicked circRNAs. The characteristic degradation products of circRNAs and their biological activities remain unclear. Therefore, developing methods to identify target circRNAs and non-target components and investigating degradation patterns will be beneficial to gaining an in-depth understanding of the properties and quality control of circRNAs vaccines. The reversed-phase HPLC (RP-HPLC) method was established for identification of target circRNAs, product-related substances, and impurities. Subsequently, we investigated the degradation patterns of circRNAs under thermal acceleration conditions and performed biological analysis of degradation products and linear precursors. Here, RP-HPLC method effectively identified circRNAs and nicked circRNAs. With thermal acceleration, circRNAs exhibited a “circular→nicked circRNAs→degradation products” degradation pattern. Biological analysis revealed that the immunogenicity of degradation products significantly decreased, whereas linear precursors did not possess immunogenicity. Thus, our established RP-HPLC method can be used for purity analysis of circRNA vaccines, which contributes to the quality control of circRNA vaccines and promoting the development of circRNA technology.

INTRODUCTION:The mRNA vaccine technologies have progressed rapidly in recent years. The COVID-19 pandemic has accelerated the application of mRNA vaccines, with research and development and clinical trials underway for many vaccines. Application of the quality by design (QbD) framework to mRNA vaccine development and establishing standardized quality control protocols for mRNA vaccines are essential for the continued development of high-quality mRNA vaccines.AREAS COVERED:mRNA vaccines include linear mRNA, self-amplifying mRNA, and circular RNA vaccines. This article summarizes the progress of research on quality control of these three types of vaccines and presents associated challenges and considerations.EXPERT OPINION:Although there has been rapid progress in research on linear mRNA vaccines, their degradation patterns remain unclear. In addition, standardized assays for key impurities, such as residual dsRNA and T7 RNA polymerase, are still lacking. For self-amplifying mRNA vaccines, a key focus should be control of stability in vivo and in vitro. For circular RNA vaccines, standardized assays, and reference standards for determining degree of circularization should be established and optimized.