Rapid identification of A29L antibodies based on mRNA immunization and high-throughput single B cell sequencing to detect Monkeypox virus.

With the large number of atypical cases in the mpox outbreak, which was classified as a global health emergency by the World Health Organization (WHO) on 23 July 2022, rapid diagnosis of mpox and diseases with similar symptoms to mpox such as chickenpox and respiratory infectious diseases in the early stages of viral infection is key to controlling the spread of the outbreak. In this study, antibodies against the monkeypox virus A29L protein were efficiently and rapidly identified by combining rapid mRNA immunization with high-throughput sequencing of individual B cells. We obtained eight antibodies with a high affinity for A29L validated by ELISA, which were was used as the basis for developing an ultrasensitive fluorescent immunochromatographic assay based on multilayer quantum dot nanobeads (SiTQD-ICA). The SiTQD-ICA biosensor utilizing M53 and M78 antibodies showed high sensitivity and stability of detection: A29L was detected within 20 min, with a minimum detection limit of 5 pg/mL. A specificity test showed that the method was non-cross-reactive with chickenpox or common respiratory pathogens and can be used for early and rapid diagnosis of monkeypox virus infection by antigen detection. This antibody identification method can also be used for rapid acquisition of monoclonal antibodies in early outbreaks of other infectious diseases for various studies.

Dendritic Cell-Mimicking Nanoparticles Promote mRNA Delivery to Lymphoid Organs.

Spleen and lymphoid organs are important targets for messenger RNA (mRNA) delivery in various applications. Current nanoparticle delivery methods rely on drainage to lymph nodes from intramuscular or subcutaneous injections. In difficult-to-transfect antigen-presenting cells (APCs), such as dendritic cells (DCs), effective mRNA transfection remains a significant challenge. In this study, a lymphatic targeting carrier using DC membranes is developed, that efficiently migrated to lymphoid organs, such as the spleen and lymph nodes. The nanoparticles contained an ionizable lipid (YK009), which ensured a high encapsulation efficacy of mRNA and assisted mRNA with endosomal escape after cellular uptake. Dendritic cell-mimicking nanoparticles (DCMNPs) showed efficient protein expression in both the spleen and lymph nodes after intramuscular injections. Moreover, in immunized mice, DCMNP vaccination elicited Spike-specific IgG antibodies, neutralizing antibodies, and Th1-biased SARS-CoV-2-specific cellular immunity. This work presents a powerful vaccine formula using DCMNPs, which represents a promising vaccine candidate for further research and development.

Monkeypox virus quadrivalent mRNA vaccine induces immune response and protects against vaccinia virus.

Monkeypox has been declared a public health emergency by the World Health Organization. There is an urgent need for efficient and safe vaccines against the monkeypox virus (MPXV) in response to the rapidly spreading monkeypox epidemic. In the age of COVID-19, mRNA vaccines have been highly successful and emerged as platforms enabling rapid development and large-scale preparation. Here, we develop two MPXV quadrivalent mRNA vaccines, named mRNA-A-LNP and mRNA-B-LNP, based on two intracellular mature virus specific proteins (A29L and M1R) and two extracellular enveloped virus specific proteins (A35R and B6R). By administering mRNA-A-LNP and mRNA-B-LNP intramuscularly twice, mice induce MPXV specific IgG antibodies and potent vaccinia virus (VACV) specific neutralizing antibodies. Further, it elicits efficient MPXV specific Th-1 biased cellular immunity, as well as durable effector memory T and germinal center B cell responses in mice. In addition, two doses of mRNA-A-LNP and mRNA-B-LNP are protective against the VACV challenge in mice. And, the passive transfer of sera from mRNA-A-LNP and mRNA-B-LNP-immunized mice protects nude mice against the VACV challenge. Overall, our results demonstrate that mRNA-A-LNP and mRNA-B-LNP appear to be safe and effective vaccine candidates against monkeypox epidemics, as well as against outbreaks caused by other orthopoxviruses, including the smallpox virus.

Improving timely treatment with a stroke emergency map: The case of northern China.

OBJECTIVE: The Chinese stroke emergency map (SEM) was implemented in 2017 to reduce prehospital and hospital delays for acute ischemic stroke (AIS) patients suitable for intravenous recombinant tissue plasminogen activator (rt-PA) thrombolysis. However, data on the time delay following the implementation of an SEM in China are limited. METHODS: Data for suspected stroke patients from the SEM registry center of Taiyuan, Shanxi Province, from August 2017 to July 2019, patients' characteristics, thrombolysis rate, and functional outcome at 90 days were analyzed. RESULTS: One thousand seven hundred and eighty six patients who arrived at hospitals within 4.5 hr of onset were included; 35.9% arrived by emergency medical services (EMSs), and 1,207 (67.6%) of the population received intravenous rt-PA. As a result of the SEM, the number of patients treated with rt-PA increased from 63.9% in phase 1 (August 2017 to July 2018) to 70.5% in phase 2 (August 2018 to July 2019). The median onset-to-door and onset-to-needle times decreased by five minutes (100 [IQR: 62-135] vs. 105 [IQR: 70-145], p = .005) and nine minutes (158 [IQR: 124-197] vs. 167 [IQR: 132-214], p = .001), respectively. Patients in phase 2 achieved greater independent function outcome at 90 days (79.9% vs. 72.1%; adjusted odds ratio, 2.010; 95% confidence interval, 1.444-2.798). The binary logistic regression models revealed that shorter onset-to-needle time (OR: 0.994; 95% CI: 0.992-0.997; p < .001) and lower baseline NIHSS scores (OR: 39.120; 95% CI: 23.477-65.188; p < .001 and OR: 18.324; 95% CI: 11.425-29.388; p < .001 and OR: 3.123; 95% CI: 2.044-4.773; p < .001) were significant predictors for the independent function outcome. CONCLUSION: The implementation of a stroke emergency map is more likely to reduce prehospital delays and improve function outcomes. Future efforts should attempt to increase EMS usage.