The Current State and Progress of Mpox Vaccine Research.

On July 23, 2022, the World Health Organization (WHO) declared the monkeypox (mpox) outbreak a "Public Health Emergency of International Concern." Since 2022, outbreaks of mpox in many countries around the world have primarily resulted in fatalities among immunocompromised individuals, such as untreated HIV/AIDS patients. Since the eradication of smallpox was declared by the WHO in 1980, the global vaccination against smallpox has been gradually discontinued. China also stopped routine smallpox vaccination in 1981. The protective effect of the smallpox vaccine has decreased over time due to aging and declining immunity in those who were vaccinated. For individuals, timely vaccination against smallpox is an effective means of protection against mpox. However, due to safety concerns with the smallpox vaccine and the limitations of current mpox vaccines, there is no vaccine that is safe, effective, and has low side effects applied in clinical settings. This article provides a comprehensive review of the development of mpox virus (MPXV) vaccines, their application in special populations, and the current state of vaccine research, considering the etiology, transmission, and prevention of the MPXV. Vaccination, as an effective method of epidemic prevention, can provide long-term immune protection and effectively reduce the severity of infection. However, as there is no licensed specific MPXV vaccine available globally, the vaccines currently used for mpox prevention are mostly smallpox vaccines. These smallpox vaccines can offer some degree of protection against mpox by activating cross-protection in the body.

Detection of monkeypox virus based on a convenient and sensitive single-step RPA-CRISPR/Cas12a strategy.

The global outbreak of monkeypox virus (MPXV) has highlighted the need for rapid molecular diagnostics techniques. In this study, a single-step recombinase polymerase amplification (RPA)-CRISPR/Cas12a system was developed for rapid and sensitive detection of MPXV. The limit of detection of this assay was 1 copy per µL of extracted nucleic acids. A heating lysis method was integrated to further simplify the sample processing workflow and shorten the assay time to 40 min from sample to result. The reaction mixture can be lyophilized to improve its accessibility in resource-limited settings. The analysis results of the proposed single-step RPA-CRISPR/Cas12a assay for clinical MPXV positive and negative samples were 100% consistent with standard PCR assay. These results demonstrate the feasibility and efficiency of this method for rapid and accurate MPXV detection in real-world settings, showcasing its potential utility in urgent and practical settings.

Ultrasensitive single-step CRISPR detection of monkeypox virus in minutes with a vest-pocket diagnostic device.

The emerging monkeypox virus (MPXV) has raised global health concern, thereby highlighting the need for rapid, sensitive, and easy-to-use diagnostics. Here, we develop a single-step CRISPR-based diagnostic platform, termed SCOPE (Streamlined CRISPR On Pod Evaluation platform), for field-deployable ultrasensitive detection of MPXV in resource-limited settings. The viral nucleic acids are rapidly released from the rash fluid swab, oral swab, saliva, and urine samples in 2 min via a streamlined viral lysis protocol, followed by a 10-min single-step recombinase polymerase amplification (RPA)-CRISPR/Cas13a reaction. A pod-shaped vest-pocket analysis device achieves the whole process for reaction execution, signal acquisition, and result interpretation. SCOPE can detect as low as 0.5 copies/µL (2.5 copies/reaction) of MPXV within 15 min from the sample input to the answer. We validate the developed assay on 102 clinical samples from male patients / volunteers, and the testing results are 100% concordant with the real-time PCR. SCOPE achieves a single-molecular level sensitivity in minutes with a simplified procedure performed on a miniaturized wireless device, which is expected to spur substantial progress to enable the practice application of CRISPR-based diagnostics techniques in a point-of-care setting.

Profiling of viral load, antibody and inflammatory response of people with monkeypox during hospitalization: a prospective longitudinal cohort study in China.

BACKGROUND: The dynamics of viral shedding and the specific humoral response against monkeypox virus (MPXV) have not been well characterized in patients across their disease course during hospitalisation. The aim of this study was to determine the viral load and the levels of antibodies against MPXV using longitudinal paired-collected samples from hospitalized patients. METHODS: Patients who were hospitalised with mpox were recruited at Beijing Ditan Hospital Capital Medical University in China between June 2 and September 23, 2023. Paired samples, including samples from skin lesions, the oropharynx, saliva, faeces, urine, plasma, and serum, were serially collected at days 1, 3, 7, and 14 after admission until discharge. Not all of the patients had samples obtained at all of the timepoints. All the samples were analysed via quantitative PCR. Virus isolation was performed by using clinical samples and Vero cells. The presence of IgM, IgA, IgG, and neutralising antibodies (NAbs) against MPXV was evaluated. The first collected plasma sample was taken when the patient was hospitalised, and the levels of cytokines and chemokines were measured in the sample. The demographic data, smallpox vaccination status, history of known exposure to MPVX, HIV status and other clinical data were collected using a standard case report form. FINDINGS: A total of 510 specimens were serially collected from 39 recruited people with mpox. Among all the samples, the skin lesions had the highest viral DNA detection rates and viral loads, and the saliva samples had the second highest rates and viral loads. One day before discharge, 85% of the dry scrabs (median Ct 28.2, range 19.0-38.3) and 70% of the saliva samples (median Ct 32.4, range 24.5-38.1) were positive for viral DNA, Of which, 23.1% of dry scrabs were positive in viral culture. The rate of viral DNA detection in the oropharyngeal, saliva, and faecal samples decreased with time, while the rates in the plasma, serum, and urine samples increased quickly before 10 days post symptom onset (PSO). The median days of appearance of MPXV-IgM, MPXV-IgA, MPXV-IgG, and NAb were at 8 (interquartile range [IQR] 7-9), 9 (7-10), 12 (9-15), and 12 (9-15) PSO, respectively. The IgM, IgA, IgG, and NAb titres increased with time. Between days 11 and 21 PSO, the NAb titres were lower in people living with HIV (PWH) than in people living without HIV (PWOH). Increased NAb titres were associated with decreased viral loads in the saliva (r = 0.28, p = 0.025), faeces (r = 0.35, p = 0.021), plasma (r = 0.30, p = 0.0044), and serum samples (r = 0.37, p = 0.001). Compared with PWOH, PWH had higher plasma levels of MIP-1α, MIP-1ß, G-CSF, IL-4, and FGF-basic. INTERPRETATION: The high positive viral culture rate of clinical samples of patients when they are discharged from the hospital indicates that effective public health management strategies are needed for people with mpox. The low NAb titres and high levels of cytokines in PWH shows that earlier treatment is needed to control inflammation in high-risk populations. FUNDING: National Natural Science Foundation of China, Chinese Academy of Medical Sciences, Fundamental Research Funds for the Central Universities for Peking Union Medical College, National Key R&D Program of China.

Comparing sputum microbiota characteristics between severe and critically ill influenza patients.

Background: Currently, limited attention has been directed toward utilizing clinical cohorts as a starting point to elucidate alterations in the lower respiratory tract (LRT) microbiota following influenza A virus (IAV) infection. Objectives: Our objective was to undertake a comparative analysis of the diversity and composition of sputum microbiota in individuals afflicted by severe and critically ill influenza patients. Methods: Sputum specimens were procured from patients diagnosed with IAV infection for the purpose of profiling the microbiota using 16S-rDNA sequencing. To ascertain taxonomic differences between the severe and critically ill influenza cohorts, we leveraged Linear Discriminant Analysis Effect Size (LEfSe). Additionally, Spearman correlation analysis was employed to illuminate associations between sputum microbiota and influenza Ct values alongside laboratory indicators. Results: Our study encompassed a total cohort of 64 patients, comprising 48 within the severe group and 16 within the critically ill group. Intriguingly, Bacteroidetes exhibited significant depletion in the critically ill cohort (p=0.031). The sputum microbiomes of the severe influenza group were hallmarked by an overrepresentation of Neisseria, Porphyromonas, Actinobacillus, Alloprevotella, TM7x, and Clostridia_UCG-014, yielding ROC-plot AUC values of 0.71, 0.68, 0.60, 0.70, 0.70, and 0.68, respectively. Notably, Alloprevotella exhibited an inverse correlation with influenza Ct values. Moreover, C-reactive protein (CRP) manifested a positive correlation with Haemophilus and Porphyromonas. Conclusion: The outcomes of this investigation lay the groundwork for future studies delving into the connection between the LRT microbiome and respiratory disorders. Further exploration is warranted to elucidate the intricate mechanisms underlying the interaction between IAV and Alloprevotella, particularly in disease progression.