A stable thymidine kinase 1 tetramer for improved quality control of serum level quantification.

DNA synthesis is a critical process for cell growth and division. In cancer patients, an enzyme called thymidine kinase 1 (TK1) is often elevated in the blood, making it a valuable biomarker for cancer diagnosis and treatment. However, previous studies have shown that recombinant TK1 can exist in unstable mixtures of tetramers and dimers, leading to inconsistent results and potentially affecting accuracy. To address this issue, we hypothesized that incorporating tetrameric coiled-coil peptides could enhance TK1 self-assembly into stable tetramers without requiring additional adenosine triphosphate. In this study, we successfully expressed a recombinant TK1 tetramer protein in the Escherichia coli system. We optimized the induction conditions, significantly increasing protein expression levels, functionality, and solubility. Size exclusion chromatography confirmed the formation of a tetrameric structure in the expressed TK1 protein, with a molecular weight of 127.2 KDa, consistent with our expectations. We also found that the TK1 tetramer exhibited higher affinity with anti-TK1 IgY than wild-type TK1, as shown by enzyme-linked immunosorbent assay experiments. Moreover, the TK1 tetramer demonstrated good stability against heating, freeze-thawing and lyophilization with almost no immunoactivity lost. These findings suggest that recombinant TK1 tetramers have the potential to serve as calibrators in diagnostic assay kits, becoming promising candidates for quality control of clinical laboratory and in vitro diagnostic reagents.

Global transboundary transmission path and risk of Mpox revealed with Least Cost Path model.

OBJECTIVES: The recent surge of Mpox outbreaks in multiple countries has garnered global attention. As of July 12, 2023, there have been 88,288 reported cases of Mpox worldwide. Although genetic variation was not found to be the cause of the epidemic outbreak, the reasons for its rapid spread remain unclear. METHODS: Using the niche method, this study identified high-risk regions for Mpox and determined that human factors are the primary contributors to global risks. To further investigate, a travel network resistance surface was created based on various modes of transportation and was combined with sea, airline, highway, and railway routes to construct the least cost path for human travel networks in different risk areas. RESULTS: The results indicated that high-risk regions for Mpox are mainly concentrated in Europe and the United States, with large risk ranges and high-risk values. The least cost path revealed three primary transmission paths rely on developed transportation networks, including internal transmission in North America, Europe-Africa, and Europe-Asia-Africa. These findings suggest that human activities, facilitated by developed travel networks, remain the main contributing factor to the spread. CONCLUSIONS: In summary, based on the Mpox epidemic report, this study conducted risk prediction and driving factor analysis on Mpox. The research results indicate that human use of transportation for long-distance activities is a key factor leading to the rapid spread of the virus. Subsequently, we focused on studying the global transmission pathways of Mpox and revealed several transmission pathways with high global population migration rates by constructing the LCPs between different high-risk areas. This study also emphasizes the importance of applying early monitoring data of Mpox to model risk prediction in controlling emerging infectious diseases, providing a new perspective for controlling Mpox and similar diseases.

A rapid and sensitive fluorescent chromatography with cloud system for MPXV point-of-care diagnosis.

Monkeypox (mpox) is spreading around the world, and its rapid diagnosis is of great significance. In the present study, a rapid and sensitive fluorescent chromatography assisted with cloud system was developed for point-of-care diagnosis of mpox. To screen high affinity antibodies, nanoparticle antigen AaLS-A29 was generated by conjugating A29 onto scaffold AaLS. Immunization with AaLS-A29 induced significantly higher antibody titers and monoclonal antibodies were generated with the immunized mice. A pair of monoclonal antibodies, MXV 14 and MXV 15, were selected for fluorescence chromatography development. The Time-Resolved Fluorescence Immunoassay (TRFIA) was used to develop the chromatography assay. After optimization of the label and concentration of antibodies, a sensitive TRFIA assay with detection limit of 20 pg/mL and good repeatability was developed. The detection of the surrogate Vaccinia virus (VACA) strain Tian Tan showed that the TRFIA assay was more sensitive than the SYBR green I based quantitative PCR. In real samples, the detection result of this assay were highly consistent with the judgement of Quantitative Real-Time PCR (Concordance Rate = 90.48%) as well as the clinical diagnosis (Kappa Value = 0.844, P < 0.001). By combining the portable detection and online cloud system, the detection results could be uploaded and shared, making this detection system an ideal system for point-of-care diagnosis of mpox both in field laboratory and outbreak investigation.

Development and Immunological Evaluation of a Multiantigen Thermostable Nanovaccine Adjuvanted with T-Cell-Activating Scaffold for African Swine Fever.

African swine fever is an acute and highly contagious infectious disease with a mortality rate of up to 100%. The lack of commercial vaccines and drugs is a serious economic threat to the global pig industry. Cell-mediated immunity plays an essential role in protection against viral infection. We previously reported the rational design of a T-cell-activating thermostable scaffold (RPT) for antigen delivery and improved cellular immunity. We conjugated antigens P30, P54, P72, CD2 V, and CP312R to RPT, using a SpyCatcher/SpyTag covalent attachment strategy to construct nanovaccines (multiantigens-RPT). Multiantigens-RPT exhibited significantly higher thermal, storage, and freeze-thaw stability. The specific antibodies IgG and IgG2a of the multiantigen-RPT-immunized were higher than the antigens cocktail-immunized by approximately 10-100 times. ELISpot demonstrated that more IFN-γ-secreting cells were produced by the multiantigen-RPT-immunized than by the antigens cocktail-immunized. Delivery of the multiantigen nanovaccine by a T-cell-activating scaffold induced strong humoral and cellular immune responses in mice and pigs and is a potentially useful candidate vaccine for the African swine fever virus.

Rapid and highly potent humoral responses to mpox nanovaccine candidates adjuvanted by thermostable scaffolds.

Monkeypox (mpox) is a zoonotic disease caused by monkeypox virus (MPXV) of the orthopoxvirus genus. The emergence and global spread of mpox in 2022 was declared as a public health emergency by World Health Organization. This mpox pandemic alarmed us that mpox still threaten global public health. Live vaccines could be used for immunization for this disease with side effects. New alternative vaccines are urgently needed for this re-emerging disease. Specific antibody responses play key roles for protection against MPXV, therefore, vaccines that induce high humoral immunity will be ideal candidates. In the present study, we developed thermostable nanovaccine candidates for mpox by conjugating MPXV antigens with thermostable nanoscafolds. Three MPXV protective antigens, L1, A29, and A33, and the thermostable Aquafex aeolicus lumazine synthase (AaLS), were expressed in E. coli and purified by Ni-NTA methods. The nanovaccines were generated by conjugation of the antigens with AaLS. Thermal stability test results showed that the nanovaccines remained unchanged after one week storage under 37℃ and only partial degradation under 60℃, indicating high thermostability. Very interesting, one dose immunization with the nanovaccine could induce high potent antibody responses, and two dose induced 2-month high titers of antibodes. In vitro virus neutralization test showed that nanovaccine candidates induced significantly higher levels of neutralization antibodies than monomers. These results indicated that the AaLS conjugation nanovaccines of MPXV antigens are highly thermostable in terms of storage and antigenic, being good alternative vaccine candidates for this re-emerging disease.

Potent immune responses against thermostable Foot-and-Mouth disease virus VP1 nanovaccine adjuvanted with polymeric thermostable scaffold.

Foot-and-mouth disease (FMD) is an acute zoonosis causes significant economic losses. Vaccines able to stimulate efficient protective immune responses are urgently needed. In this study, Escherichia coli-derived recombinant VP1 of serotype A and O FMD virus (FMDV) was conjugated to thermostable scaffold lumazine synthase (LS) or Quasibacillus thermotolerans encapsulin (QtEnc) using a robust plug-and-display SpyTag/SpyCatcher system to generate multimeric nanovaccines. These nanovaccines induced highly potent antibody responses in vaccinated mice. On day 14 after the first immunisation, antibody titres were approximately 100 times higher than those of monomer antigens. Both vaccines induced high and long-term IgG antibody production. Moreover, the QtEnc-VP1 nanovaccine induced higher antibody titres than the LS-VP1 nanovaccine. The nanovaccines also induced Th1-biased immune responses and higher levels of neutralising antibodies. These data indicated that FMDV nanovaccines generated by conjugating VP1 with a thermostable scaffold are highly immunogenic and ideal candidates for FMDV control in low-resource areas.

Driving effect of multiplex factors on Mpox in global high-risk region, implication for Mpox based on one health concept.

Mpox is an ongoing viral zoonotic disease epidemic worldwide. Being different from conventional animal-to-human transmission, the present outbreak is mainly caused by human-to-human transmission of Mpox virus, putting forward the risk of worldwide epidemic. The current spatial distribution characteristics and risk area prediction are urgently needed for preparedness for prevention and control of the disease based on the One Health strategy. In the present study, the global outbreak point of Mpox virus were collected and used to predict potential global risk of Mpox virus with ecological niche model constructed with a combination of eco-geographical, anthropoid, meteorological, and host variables. The results showed that human factors are the key to the risk and prevalence of Mpox. The risk map indicated that Mpox may affect extensive areas worldwide. Europe and North America have the highest risk of Mpox. Although most areas have never recorded Mpox before, there are some high-risk areas in Asia. Our findings highlight population density is the most important contributing factor for high-risk area. Many large cities with dense populations, developed transportation, and high migration rate in the world, are in high risks. At present, the spread of Mpox is highly valued in the world and strict prevention and control measures have been taken. However, under the influence of human factors, Mpox has the potential of a global pandemic. The risk area prediction and main risk factors provide key information for targeted preparedness for prevention and control of Mpox outbreak and avoiding potential global epidemic through the One Health approach.

Thermostable T Cell Multiepitope Nanoparticle Antigens Inducing Potent Immune Responses against the Swine Fever Virus.

African swine fever (ASF) is caused by the African swine fever virus (ASFV) and is a highly contagious, acute, febrile disease that has high morbidity and mortality rates in domestic and wild swine. However, a safe and effective vaccine against ASF remains unavailable as single antigens fail to provide sufficient protection. Therefore, a combination of multiple antigens with an efficient delivery system might be an alternative strategy. Herein, a de novo-designed antigen with multiple T-cell epitopes (TEPs) of ASFV was conjugated for surface display on self-assembled nanoparticles (NPs) of Aquifex aeolicus lumazine synthase (AaLS) and Quasibacillus thermotolerans encapsulin (QT) through the SpyCatcher/SpyTag system to construct nanovaccines (TEP-Spy-NPs). TEP-Spy-NPs exhibited significantly more thermal, storage, and freeze-thaw stability in comparison to TEP monomers. TEP-Spy-NPs were highly immunogenic and induced strong polyclonal antibody responses in mice and pigs. The specific antibody titers against the TEP of the TEP-Spy-AaLS and TEP-Spy-QT groups were significantly higher than those of the TEP monomer immune group after the second booster immunization. The antibody titer against TEP of the TEP-Spy-QT group was approximately twice that of the TEP-Spy-AaLS group in mice. ELISpot analysis demonstrated that more IFN-γ- and IL-2-secreting splenic lymphocytes were produced by TEP-Spy-AaLS- and TEP-Spy-QT-immunized mice than by TEP monomer-immunized mice. TEP-Spy-NPs elicited stronger cellular immunity and in vivo immunity in immunized pigs than did TEP monomers. Thus, the TEP nanovaccine successfully induced strong humoral and cellular immune responses in mice and pigs, and TEP-Spy-NPs have the potential as candidate vaccines for ASFV.

Factors associated with the utilization of diagnostic tools among countries with different income levels during the COVID-19 pandemic.

BACKGROUND: Disparities in the utilization of essential medical products are a key factor contributing to inequality in health outcomes. We aimed to analyze the trends and influencing factors in using Coronavirus disease 2019 (COVID-19) diagnostic tools and disparities in countries with different income levels. METHODS: We conducted a cross-sectional study using open and publicly available data sources. Data were mainly collected from the Foundation for Innovative New Diagnostics, "Our World in Data," and the Global Burden of Disease databases. Negative binomial regression model and generalized linear mixed model were employed to investigate into five sets of factors associated with the usage of diagnostics: severity of COVID-19, socioeconomic status, health status, medical service capacity, and rigidity of response. Dominance analysis was utilized to compare the relative importance of these factors. The Blinder-Oaxaca decomposition was used to decompose the difference in the usage of diagnostics between countries. RESULTS: The total COVID-19 testing rate ranged from 5.13 to 22,386.63 per 1000 people from March 2020 to October 2022 and the monthly testing rate declined dramatically from January 2022 to October 2022 (52.37/1000 vs 5.91/1000).. The total testing rate was primarily associated with socioeconomic status (37.84%), with every 1 standard deviation (SD) increase in Gross Domestic Product per capita and the proportion of people aged ≥ 70, the total testing rate increased by 88% and 31%. And so is the medical service capacity (33.66%), with every 1 SD increase in health workforce density, the number increased by 38%. The monthly testing rate was primarily associated with socioeconomic status (34.72%) and medical service capacity (28.67%), and the severity of COVID-19 (21.09%). The average difference in the total testing rates between high-income and low-income countries was 2726.59 per 1000 people, and 2493.43 (91.45%) of the differences could be explained through the five sets of factors. CONCLUSIONS: Redoubling the efforts, such as local manufacturing, regulatory reliance, and strengthening the community health workforce and laboratory capacity in low- and middle-income countries (LMICs) cannot be more significant for ensuring sustainable and equitable access to diagnostic tools during pandemic.

Evolving market-shaping strategies to boost access to essential medical products in developing countries with HIV self-testing as a case study.

The COVID-9 pandemic has exacerbated health inequities among countries in the Global South with limited access to essential medical products, leading to a higher infection and mortality rate, especially among vulnerable populations. Despite tremendous progress in global health financing, the estimated annual financing gap in developing countries is projected to reach US$371 billion per year by 2030. Therefore, developing market-shaping strategies is of great importance in ensuring adequate supply, affordable prices, and equitable access to essential medical products in low-and middle-income countries. We propose a strategic and appropriate market-shaping intervention framework for governments, international organizations, and NGOs to maximize access to essential medical products in developing countries. In the health field, we believe that market shaping strategy could be defined as a set of purposeful activities that market forces may intervene with to advance the development, production, supply, and distribution of global goods for health, making essential medical products more affordable, accessible, innovative, sustainable and quality assured. We argue that when designing a market-shaping strategy, policy or decision-makers must take full advantage of the key drivers to keep the market dynamic, interactive, and constantly evolving to meet the unmet medical needs. In addition, different forms of market-shaping interventions are determined by objectives and specific issues to be addressed. More comprehensive market shaping strategies, including the strategic use of market expansion, market disruption, market maintenance, and market contraction alone or together, deserve to be explored and key stakeholders are also expected to join forces to make the intervention more efficient and productive.

Seasonal malaria chemoprevention in Africa and China's upgraded role as a contributor: a scoping review.

BACKGROUND: Children under five are the vulnerable population most at risk of being infected with Plasmodium parasites, especially in the Sahel region. Seasonal malaria chemoprevention (SMC) recommended by World Health Organization (WHO), has proven to be a highly effective intervention to prevent malaria. Given more deaths reported during the COVID-19 pandemic than in previous years due to the disruptions to essential medical services, it is, therefore, necessary to seek a more coordinated and integrated approach to increasing the pace, coverage and resilience of SMC. Towards this end, fully leverage the resources of major players in the global fight against malaria, such as China could accelerate the SMC process in Africa. METHODS: We searched PubMed, MEDLINE, Web of Science, and Embase for research articles and the Institutional Repository for Information Sharing of WHO for reports on SMC. We used gap analysis to investigate the challenges and gaps of SMC since COVID-19. Through the above methods to explore China's prospective contribution to SMC. RESULTS: A total of 68 research articles and reports were found. Through gap analysis, we found that despite the delays in the SMC campaign, 11.8 million children received SMC in 2020. However, there remained some challenges: (1) a shortage of fully covered monthly courses; (2) lack of adherence to the second and third doses of amodiaquine; (3) four courses of SMC are not sufficient to cover the entire malaria transmission season in areas where the peak transmission lasts longer; (4) additional interventions are needed to consolidate SMC efforts. China was certified malaria-free by WHO in 2021, and its experience and expertise in malaria elimination can be shared with high-burden countries. With the potential to join the multilateral cooperation in SMC, including the supply of quality-assured health commodities, know-how transfer and experience sharing, China is expected to contribute to the ongoing scale-up of SMC. CONCLUSIONS: A combination of necessary preventive and curative activities may prove beneficial both for targeted populations and for health system strengthening in the long run. More actions are entailed to promote the partnership and China can be one of the main contributors with various roles.

Integrative Analysis of Fruit Quality and Anthocyanin Accumulation of Plum cv. 'Cuihongli' (Prunus salicina Lindl.) and Its Bud Mutation.

Fruit color is one of the quality indicators to judge the freshness of a plum. The coloring process of plum skin is valuable for research due to the high nutritional quality of anthocyanins found in plums. 'Cuihongli' (CHL) and its precocious mutant variety 'Cuihongli Red' (CHR) were used to analyze the changes of fruit quality and anthocyanin biosynthesis during plum development. The results showed that, during the development of the two plums, the total soluble solid and soluble sugar contents were highest at the mature stage, as the titratable acid trended gradually downward as the fruits of the two cultivars matured, and the CHR fruit showed higher sugar content and lower acid content. In addition, the skin of CHR turned red in color earlier than CHL. Compared with CHL, the skin of CHR had higher anthocyanin concentrations, higher activities of phenylalanine ammonia-lyase (PAL), chalcone isomerase (CHI), dihydroflavonol-4-reductase (DFR), and UDPglucose: flavonoid-3-O-glucosyltransferase (UFGT), and higher transcript levels of genes associated with anthocyanin production. In the flesh of the two cultivars, no anthocyanin content was detected. Taken together, these results suggest that the mutation exerted a major effect on anthocyanin accumulation via modification of the level of transcription; thus, CHR advances the ripening period of 'Cuihongli' plum and improves the fruit quality.

Regulatory reliance for convergence and harmonisation in the medical device space in Asia-Pacific.

While some sort of regulatory convergence and harmonisation are often needed for achieving regulatory reliance, in reality, regulatory reliance as a strategy towards convergence and harmonisation has never been more significant in Asia-Pacific (APAC). A sustained, rapid and large-scale provision of medical devices, including articles and apparatus used in diagnosis, care, treatment or prevention of disease and softwares, etc, across national boundaries, is the key to winning the fight against future pandemics and improving people's well-being in such a populous and geographically diverse region. The COVID-19 pandemic highlighted the value of regulatory reliance to enable easier access to medical devices that have gone through regulatory approvals in countries with mature regulatory systems based on the Quality Management System and product assessment guidelines of the International Medical Device Regulators Forum. This analysis focuses on why regulatory reliance is needed, how much has been achieved, its impact on the development of the medical device industry and challenges to be addressed in the region. By drawing on the experience from the Singapore Health Sciences Authority-Thai Food and Drug Administration regulatory reliance pilot and Vietnam's inclusion of Korea Ministry of Food and Drug Safety and China National Medical Products Administration as reference markets for fast review/approval, it aims to explore next viable steps and future trend of the APAC regional regulatory harmonisation mechanism through regulatory reliance in the post-COVID-19 era.