Three in one: An effective and universal vaccine expressing heterologous tandem RBD trimer by rabies virus vector protects mice against SARS-CoV-2.

The rapid emergence of Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2) variants, coupled with severe immune evasion and imprinting, has jeopardized the vaccine efficacy, necessitating urgent development of broad protective vaccines. Here, we propose a strategy employing recombinant rabies viruses (RABV) to create a universal SARS-CoV-2 vaccine expressing heterologous tandem receptor-binding domain (RBD) trimer from the SARS-CoV-2 Prototype, Delta, and Omicron strains (SRV-PDO). The results of mouse immunization indicated that SRV-PDO effectively induced cellular and humoral immune responses, and demonstrated higher immunogenicity and broader SARS-CoV-2 neutralization compared to the recombinant RABVs that only expressed RBD monomers. Moreover, SRV-PDO exhibited full protection against SARS-CoV-2 in the challenge assay. This study demonstrates that recombinant RABV expressing tandem RBD-heterotrimer as a multivalent immunogen could elicit a broad-spectrum immune response and potent protection against SARS-CoV-2, making it a promising candidate for future human or veterinary vaccines and offering a novel perspective in other vaccine design.

Immunogenicity evaluation of a bivalent vaccine based on a recombinant rabies virus expressing gB protein of FHV-1 in mice and cats.

Feline viral rhinotracheitis (FVR) is caused by the feline herpesvirus-1 (FHV-1), which commonly results in upper respiratory symptoms, and can result in death in the kittens and weak cats. Rabies is an infectious disease with zoonotic characteristics highly relevant to public health and also poses a serious threat to cats. Vaccines are the most effective method to control the spread of both FHV-1 and RABV and have the advantage that they produce long-term specific immune responses. In this study, we constructed a bivalent vaccine against FHV-1 and rabies virus (RABV) simultaneously. The vaccine was constructed by cloning FHV-1 gB into a RABV based vector, and the recombinant RABV (SRV9-FHV-gB) expressing the FHV-1 gB protein was rescued. The growth characteristics of SRV9-FHV-gB were analyzed on NA and BSR cells. To assess the immunogenicity of the vaccine, mice and cats were immunized with SRV9-FHV-gB supplemented with Gel02 adjuvant. The SRV9-FHV-gB exhibited the same growth characteristics as the parent virus SRV9 in both BSR cells and NA cells. The safety of SRV9-FHV-gB was evaluated using 5-day-old and 14-day-old suckling mice. The results showed that mice infected with the SRV9-FHV-gB survived for longer than those in the SRV9 group. Mice immunized with inactivated SRV9-FHV-gB produced high titers of specific antibodies against FHV-1 and neutralizing antibodies against RABV. Cats that received three immunizations with SRV9-FHV-gB also produced neutralizing antibodies against both FHV-1 and RABV. This study represents the first time that a bivalent vaccine targeting FHV-1 and RABV has been constructed, laying the foundations and providing inspiration for the development of other multivalent vaccines.

Inactivated Recombinant Rabies Virus Displaying the Nipah Virus Envelope Glycoproteins Induces Systemic Immune Responses in Mice.

Nipah virus (NiV) causes severe, lethal encephalitis in humans and pigs. However, there is no licensed vaccine available to prevent NiV infection. In this study, we used the reverse genetic system based on the attenuated rabies virus strain SRV9 to construct two recombinant viruses, rSRV9-NiV-F and rSRV9-NiV-G, which displayed the NiV envelope glycoproteins F and G, respectively. Following three immunizations in BALB/c mice, the inactivated rSRV9-NiV-F and rSRV9-NiV-G alone or in combination, mixed with the adjuvants ISA 201 VG and poly (I:C), were able to induce the antigen-specific cellular and Th1-biased humoral immune responses. The specific antibodies against rSRV9-NiV-F and rSRV9-NiV-G had reactivity with two constructed bacterial-like particles displaying the F and G antigens of NiV. These data demonstrate that rSRV9-NiV-F or rSRV9-NiV-G has the potential to be developed into a promising vaccine candidate against NiV infection.

A bacterium-like particle vaccine displaying protective feline herpesvirus 1 antigens can induce an immune response in mice and cats.

Feline herpesvirus 1 (FHV-1) is a highly transmissible virus that mainly causes ocular and upper respiratory infections in cats and seriously threatens the health of domestic cats and captive or wild cats (such as tigers, cheetahs, and lions). Vaccination is crucial to reduce the incidence rate and mortality of cats infected with FHV-1. In this study, three bacterium-like particles (BLPs) displaying the gB, gC, and gD proteins of FHV-1 were constructed based on a gram-positive enhancer matrix-protein anchor (GEM-PA) surface display system. Indirect immunofluorescence assay, western blot, and electron microscopy results showed that gB, gC or gD protein of FHV-1 was successfully displayed on the surface of GEM particles. Additionally, we designed one more BLPs, designated gB&gC&gD-GEM, which consisted of a mixture of gB-GEM, gC-GEM, and gD-GEM at a protein content ratio of 1:1:1. Mice were immunized with the four BLPs mixed with Gel02 adjuvant, and the results indicated that neutralizing antibody level in the gB&gC&gD-GEM group was superior than those in the other groups. Moreover, gB&gC&gD-GEM significantly increased the secretion of cytokines, as well as the activation and maturation of B cells. It also boosted the production of central memory T cells among CD4 + and CD8 + T cells. Moreover, gB&gC&gD-GEM mixed with Gel02 adjuvant provoked an antibody response in cats. In conclusion, the BLPs vaccine prepared from gB&gC&gD-GEM induced specific humoral and cellular immune responses to FHV-1 and be used as a potential vaccine candidate for the control of FHV-1 infection in cats.

Molecular Engineering of AIE Photosensitizers for Inactivation of Rabies Virus.

Rabies is a zoonotic neurological disease caused by the rabies virus (RABV) that is fatal to humans and animals. While several post-infection treatment have been suggested, developing more efficient and innovative antiviral methods are necessary due to the limitations of current therapeutic approaches. To address this challenge, a strategy combining photodynamic therapy and immunotherapy, using a photosensitizer (TPA-Py-PhMe) with high type I and type II reactive oxygen species (ROS) generation ability is proposed. This approach can inactivate the RABV by killing the virus directly and activating the immune response. At the cellular level, TPA-Py-PhMe can reduce the virus titer under preinfection prophylaxis and postinfection treatment, with its antiviral effect mainly dependent on ROS and pro-inflammatory factors. Intriguingly, when mice are injected with TPA-Py-PhMe and exposed to white light irradiation at three days post-infection, the onset of disease is delayed, and survival rates improved to some extent. Overall, this study shows that photodynamic therapy and immunotherapy open new avenues for future antiviral research.

Benzo [a] pyrene-loaded aged polystyrene microplastics promote colonic barrier injury via oxidative stress-mediated notch signalling.

The adsorption of toxic substances on polystyrene microplastics (PSMPs) can modify their biological toxicity and exacerbate the threat to human health. The effects of benzo [a] pyrene (B (a) P)-loaded aged PSMPs on colonic barrier integrity remains unclear. Here, we showed that binding environmentally relevant concentrations of B (a) P alteredl̥ the physicochemical features and markedly enhanced the toxicity of PSMPs. Compared to pristine PSMP, PSMP@B (a) P promoted colonic barrier degradation, body weight loss, colon length shortening, oxidative stress (OS), autophagy, inflammation, and bacterial translocation. Microplastic (MP) exposure induced injury to the colon barrier, including tight junction (TJ) and mucosal barriers, via overactivation of the Notch signalling pathway under increased OS in mice and intestinal organoids. Notably, PSMP@B (a) P exposure exacerbated damage to TJ and the mucosal barrier via the overproduction of reactive oxygen species (ROS), which could be related to the release of B (a) P from PSMP@B (a) P induced by the acidic environment of autophagosomes, which in turn exert synergistic toxic effects with PSMPs. Our study elucidates some of the potential molecular mechanisms by which B (a) P enhances PSMP-related intestinal toxicity, which provides a potential therapeutic approach for diseases caused by PSMP@B (a)P and PSMP pollution.

Bif-1c Attenuates Viral Proliferation by Regulating Autophagic Flux Blockade Induced by the Rabies Virus CVS-11 Strain in N2a Cells.

Bax-interacting factor-1 (Bif-1) is a multifunctional protein involved in apoptosis, autophagy, and mitochondrial morphology. However, the associations between Bif-1 and viruses are poorly understood. As discrete Bif-1 isoforms are selectively expressed and exert corresponding effects, we evaluated the effects of neuron-specific/ubiquitous Bif-1 isoforms on rabies virus (RABV) proliferation. First, infection with the RABV CVS-11 strain significantly altered Bif-1 expression in mouse neuroblastoma (N2a) cells, and Bif-1 knockdown in turn promoted RABV replication. Overexpression of neuron-specific Bif-1 isoforms (Bif-1b/c/e) suppressed RABV replication. Moreover, our study showed that Bif-1c colocalized with LC3 and partially alleviated the incomplete autophagic flux induced by RABV. Taken together, our data reveal that neuron-specific Bif-1 isoforms impair the RABV replication process by abolishing autophagosome accumulation and blocking autophagic flux induced by the RABV CVS-11 strain in N2a cells. IMPORTANCE Autophagy can be triggered by viral infection and replication. Autophagosomes are generated and affect RABV replication, which differs by viral strain and infected cell type. Bax-interacting factor-1 (Bif-1) mainly has a proapoptotic function but is also involved in autophagosome formation. However, the association between Bif-1-involved autophagy and RABV infection remains unclear. In this study, our data reveal that a neuron-specific Bif-1 isoform, Bif-1c, impaired viral replication by unchoking autophagosome accumulation induced by RABV in N2a cells to a certain extent. Our study reveals for the first time that Bif-1 is involved in modulating autophagic flux and plays a crucial role in RABV replication, establishing Bif-1 as a potential therapeutic target for rabies.

A recombinant rabies virus chimera expressing the DC-targeting molecular MAB2560 shows enhanced vaccine immunogenicity through activation of dendritic cells.

BACKGROUND: Rabies, caused by the rabies virus (RABV), is an ancient and neglected zoonotic disease posing a large public health threat to humans and animals in developing countries. Immunization of animals with a rabies vaccine is the most effective way to control the epidemic and the occurrence of the disease in humans. Therefore, the development of cost-effective and efficient rabies vaccines is urgently needed. The activation of dendritic cells (DCs) is known to play an important role in improving the host immune response induced by rabies vaccines. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we constructed a recombinant virus, rCVS11-MAB2560, based on the reverse genetic system of the RABV CVS11 strain. The MAB2560 protein (a DC-targeting molecular) was chimeric expressed on the surface of the viral particles to help target and activate the DCs when this virus was used as inactivated vaccine. Our results demonstrated that inactivated rCVS11-MAB2560 was able to promote the recruitment and/or proliferation of DC cells, T cells and B cells in mice, and induce good immune memory after two immunizations. Moreover, the inactivated recombinant virus rCVS11-MAB2560 could produce higher levels of virus-neutralizing antibodies (VNAs) in both mice and dogs more quickly than rCVS11 post immunization. CONCLUSIONS/SIGNIFICANCE: In summary, the recombinant virus rCVS11-MAB2560 chimeric-expressing the molecular adjuvant MAB2560 can stimulate high levels of humoral and cellular immune responses in vivo and can be used as an effective inactivated rabies vaccine candidate.

[Monoterpenoids from Paeoniae Radix Rubra based on UPLC-Q-TOF-MS].

The ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used to conduct the qualitative analysis of the monoterpene chemical components from Paeoniae Radix Rubra. Gradient elution was performed on C_(18) HD(2.1 mm×100 mm, 2.5 µm) column with a mobile phase of 0.1% formic acid(A) and acetonitrile(B). The flow rate was 0.4 mL·min~(-1) and the column temperature was 30 ℃. MS analysis was conducted in both positive and negative ionization modes using electrospray ionization(ESI) source. Qualitative Analysis 10.0 was used for data processing. The identification of chemical components was realized by the combination of standard compounds, fragmentation patterns, and mass spectra data reported in the literature. Forty-one monoterpenoids in Paeoniae Radix Rubra extract were identified. Among them, 8 compounds were reported in Paeoniae Radix Rubra for the first time and 1 was presumed to be the new compound 5″-O-methyl-galloylpaeoniflorin or its positional isomer. The method in this study realizes the rapid identification of monoterpenoids from Paeoniae Radix Rubra and provides a material and scientific basis for quality control and further study on the pharmaceutical effect of Paeoniae Radix Rubra.

[Analysis on quality value transmitting of Qinggusan reference sample of classical prescription based on quantitative fingerprint technology].

Qinggusan is the 69th prescription in the first batch of "Catalogue of Ancient Chinese Classic Formulas". In modern clinical practice， Qinggusan is mainly used to treat noninfectious fever. However， because few studies on Qinggusan reference samples and their quality value transfer are available， the development and promotion of its compound preparations are restricted. Therefore， establishing an accurate and comprehensive quality control method to clarify the critical quality attributes of Qinggusan reference samples is of great importance. In this study， 15 batches of Qinggusan reference samples were processed to determine the range of their dry extract ratios. Quantitative high-performance liquid chromatography （HPLC） fingerprint analysis was performed using a Waters Symmetry Shield RP18 column （250 mm×4.6 mm， 5 µm） with acetonitrile-0.1% （v/v） formic acid aqueous solution as the mobile phase in gradient elution mode. The flow rate was 1.0 mL/min， the column temperature was 30 ℃， and the detection wavelength was 254 nm. The HPLC fingerprints of the Qinggusan reference samples were established under these conditions to evaluate their similarity. The established method was systematically validated and found to demonstrate good precision， repeatability， and sample stability. Subsequently， characteristic peaks were identified and attributed by HPLC-quadrupole-time-of-flight-mass spectrometry （HPLC-Q-TOF-MS） analysis. MS was performed in electrospray ionization mode， the data were collected in both positive- and negative-ion modes， and the detection range was m/z 50-2000. The contents and transfer rate ranges of the index components， namely， gentiopicrin， mangiferin， picroside Ⅱ， picroside Ⅰ， and glycyrrhizic acid， were determined to analyze the quality value transfer of the samples. The results demonstrated that the dry extract rate of the 15 batches of Qinggusan reference samples ranged from 24.10% to 26.88% and that their fingerprint similarities were generally greater than 0.95. Twelve common peaks were identified by reference identification， literature comparison， and high-resolution MS analysis. Twelve compounds， including six iridoid glycosides， two flavonoids， one alkaloid， one triterpenoid saponin， and two others. Among them， L-picein， androsin， picroside Ⅳ， picroside Ⅱ and picroside Ⅰ were from Picrorhizae Rhizoma， loganin acid， swertiamarin and gentiopicrin were from Gentianae Macrophyllae Radix， neomangiferin and mangiferin were from Anemarrhenae Rhizoma， dichotomine B was from Stellariae Radix， and glycyrrhizic acid was from Glycyrrhizae Radix et Rhizoma. The five key components presented good linear relationships in their respective linear ranges， and all correlation coefficients were higher than 0.999. The relative standard deviations （RSDs） of precision， stability， and repeatability were less than 1.3%. The average recoveries varied between 95.92% and 102.5%， with RSDs less than 3.9%； these values meet the requirements defined in the 2020 edition of the Chinese Pharmacopoeia. The contents of gentiopicrin， mangiferin， picroside Ⅱ， picroside Ⅰ， and glycyrrhizic acid in the 15 batches of reference samples were in the range of 17.92-27.55， 1.83-4.42， 23.08-36.44， 8.43-15.04， and 0.94-2.39 mg/g， respectively， and their transfer rates from the decoction pieces to the reference samples were 47.91%-63.95%， 22.96%-59.39%， 60.82%-77.82%， 64.25%-99.53%， and 15.30%-39.30%， respectively. In this study， the chemical components of Qinggusan reference samples were comprehensively identified and their quality value transfer was studied through the combination of HPLC fingerprinting and MS. Clarification of the critical quality attributes of Qinggusan reference samples could provide a basis for the quality control of Qinggusan compound preparations.

An inactivated recombinant rabies virus chimerically expressed RBD induces humoral and cellular immunity against SARS-CoV-2 and RABV.

Many studies suggest that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect various animals and transmit among animals, and even to humans, posing a threat to humans and animals. There is an urgent need to develop inexpensive and efficient animal vaccines to prevent and control coronavirus disease 2019 (COVID-19) in animals. Rabies virus (RABV) is another important zoonotic pathogen that infects almost all warm-blooded animals and poses a great public health threat. The present study constructed two recombinant chimeric viruses expressing the S1 and RBD proteins of the SARS-CoV-2 Wuhan01 strain based on a reverse genetic system of the RABV SRV9 strain and evaluated their immunogenicity in mice, cats and dogs. The results showed that both inactivated recombinant viruses induced durable neutralizing antibodies against SARS-CoV-2 and RABV and a strong cellular immune response in mice. Notably, inactivated SRV-nCoV-RBD induced earlier antibody production than SRV-nCoV-S1, which was maintained at high levels for longer periods. Inactivated SRV-nCoV-RBD induced neutralizing antibodies against both SARS-CoV-2 and RABV in cats and dogs, with a relatively broad-spectrum cross-neutralization capability against the SARS-CoV-2 pseudoviruses including Alpha, Beta, Gamma, Delta, and Omicron, showing potential to be used as a safe bivalent vaccine candidate against COVID-19 and rabies in animals.

Rapid and Visual Detection of SARS-CoV-2 RNA Based on Reverse Transcription-Recombinase Polymerase Amplification with Closed Vertical Flow Visualization Strip Assay.

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was initially identified in 2019, after which it spread rapidly throughout the world. With the progression of the epidemic, new variants of SARS-CoV-2 with faster transmission speeds and higher infectivity have constantly emerged. The proportions of people asymptomatically infected or reinfected after vaccination have increased correspondingly, making the prevention and control of COVID-19 extremely difficult. There is therefore an urgent need for rapid, convenient, and inexpensive detection methods. In this paper, we established a nucleic acid visualization assay targeting the SARS-CoV-2 nucleoprotein (N) gene by combining reverse transcription-recombinase polymerase amplification with closed vertical flow visualization strip (RT-RPA-VF). This method had high sensitivity, comparable to that of reverse transcription-quantitative PCR (RT-qPCR), and the concordance between RT-RPA-VF and RT-qPCR methods was 100%. This detection method is highly specific and is not compatible with bat coronavirus HKU4, human coronaviruses 229E, OC43, and HKU1-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), or other respiratory pathogens. However, multiple SARS-CoV-2 variants are detectable within 25 min at 42°C using this visual method, including RNA transcripts of the Wuhan-Hu-1 strain at levels as low as 1 copy/µL, the Delta strain at 1 copy/µL, and the Omicron strain at 0.77 copies/µL. The RT-RPA-VF method is a simple operation for the rapid diagnosis of COVID-19 that is safe and free from aerosol contamination and could be an affordable and attractive choice for governments seeking to promote their emergency preparedness and better their responses to the continuing COVID-19 epidemic. In addition, this method also has great potential for early monitoring and warning of the epidemic situation at on-site-nursing points. IMPORTANCE The global COVID-19 epidemic, ongoing since the initial outbreak in 2019, has caused panic and huge economic losses worldwide. Due to the continuous emergence of new variants, COVID-19 has been responsible for a higher proportion of asymptomatic patients than the previously identified SARS and MERS, which makes early diagnosis and prevention more difficult. In this manuscript, we describe a rapid, sensitive, and specific detection tool, RT-RPA-VF. This tool provides a new alternative for the detection of SARS-CoV-2 variants in a range as low as 1 to 0.77 copies/µL RNA transcripts. RT-RPA-VF has great potential to ease the pressure of medical diagnosis and the accurate identification of patients with suspected COVID-19 at point-of-care.

Treat the "Untreatable" by a Photothermal Agent: Triggering Heat and Immunological Responses for Rabies Virus Inactivation.

Rabies is a fatal neurological zoonotic disease caused by the rabies virus (RABV), and the approved post-exposure prophylaxis (PEP) procedure remains unavailable in areas with inadequate medical systems. Although strategies have been proposed for PEP and postinfection treatment (PIT), because of the complexity of the treatment procedures and the limited curative outcome, developing an effective treatment strategy remains a holy grail in rabies research. Herein, a facile approach is proposed involving photothermal therapy (PTT) and photothermally triggered immunological effects to realize effective PEP and PIT simultaneously. The designed photothermal agent (N+ TT-mCB nanoparticles) featured positively charged functional groups and high photo-to-heat efficiency, which are favorable for virus targeting and inactivation. The level of the virus at the site of infection in mice is significantly decreased upon treatment with orthotopic PTT, and the transfer of the virus to the brain is significantly inhibited. Furthermore, the survival ratio of the mice three days postinfection is increased by intracranial injection of N+ TT-mCB and laser irradiation. Overall, this work provides a platform for the effective treatment of RABV and opens a new avenue for future antiviral studies.

A rabies virus-vectored vaccine expressing two copies of the Marburg virus glycoprotein gene induced neutralizing antibodies against Marburg virus in humanized mice.

Marburg virus disease (MVD) is a lethal viral haemorrhagic fever caused by Marburg virus (MARV) with a case fatality rate as high as 88%. There is currently no vaccine or antiviral therapy approved for MVD. Due to high variation among MARV isolates, vaccines developed against one strain fail to protect against other strains. Here we report that three recombinant rabies virus (RABV) vector vaccines encoding two copies of GPs covering both MARV lineages induced pseudovirus neutralizing antibodies in BALB/c mice. Furthermore, high-affinity human neutralizing antibodies were isolated from a humanized mouse model. The three vaccines produced a Th1-biased serological response similar to that of human patients. Adequate sequential immunization enhanced the production of neutralizing antibodies. Virtual docking suggested that neutralizing antibodies induced by the Angola strain seemed to be able to hydrogen bond to the receptor-binding site (RBS) in the GP of the Ravn strain through hypervariable regions 2 (CDR2) and CDR3 of the VH region. These findings demonstrate that three inactivated vaccines are promising candidates against different strains of MARV, and a novel fully humanized neutralizing antibody against MARV was isolated.

Self-cleaning wearable masks for respiratory infectious pathogen inactivation by type I and type II AIE photosensitizer.

Although face masks as personal protective equipment (PPE) are recommended to control respiratory diseases with the on-going COVID-19 pandemic, improper handling and disinfection increase the risk of cross-contamination and compromise the effectiveness of PPE. Here, we prepared a self-cleaning mask based on a highly efficient aggregation-induced emission photosensitizer (TTCP-PF6) that can destroy pathogens by generating Type I and Type II reactive oxygen species (ROS). The respiratory pathogens, including influenza A virus H1N1 strain and Streptococcus pneumoniae (S. pneumoniae) can be inactivated within 10 min of ultra-low power (20 W/m2) white light or simulated sunlight irradiation. This TTCP-PF6-based self-cleaning strategy can also be used against other airborne pathogens, providing a strategy for dealing with different microbes.

[Quality evaluation of Fufang Jinqiancao granules based on ultra performance liquid chromatography-ultraviolet detection quantitative fingerprint combined with chemical pattern recognition].

Fufang Jinqiancao granules have a large market demand due to the fact that they contain diuretics, inhibit urinary calculi formation, and exhibit both anti-inflammatory and antioxidant effects. In the current study, a fast and efficient quantitative ultra performance liquid chromatography-ultraviolet detection (UPLC-UV) fingerprinting method was established to analyze the Fufang Jinqiancao granules, while a chemical pattern recognition technology was used to evaluate the quality of the granules over different years. More specifically, the UPLC-UV system consisted of a Waters Acquity UPLC BEH C18 column (100 mm×2.1 mm, 1.7 µm), acetonitrile (mobile phase A), and a 0.1% formic acid aqueous solution (mobile phase B), wherein a gradient elution protocol was followed. Ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS, Agilent Infinity Ⅱ 1290-6545) was used in combination with reference substances and literature comparisons to identify the common peaks present in the quantitative fingerprint. The fingerprints of 35 batches of Fufang Jinqiancao granules were established by means of the quantitative UPLC-UV fingerprinting method, and the fingerprint data obtained for these samples were further analyzed by chemical pattern recognition techniques, including hierarchical cluster analysis (HCA) and principal component analysis (PCA). The quality difference markers, namely mangiferin and isomangiferin, were screened, and their contents were determined. It was found that 12 common peaks existed in the fingerprint of the Fufang Jinqiancao granules, and the similarities of all 35 batches of samples were greater than 0.952. In addition, for the purpose of HCA, the 35 batches were divided into two categories, of which sample years 2018 and 2019 belonged to one category, and sample years 2020 and 2021 belonged to another category. Notably, PCA gave the same clustering trends as HCA. Based on the obtained results, the mangiferin and isomangiferin components responsible for the differences between the 2018, 2019 and 2020, 2021 samples were further screened by orthogonal partial least squares discriminant analysis (OPLS-DA). Moreover, the contents of the 35 batches of samples were determined using the two differential markers mangiferin and isomangiferin as indicators. The obtained results indicated that the chromatographic peaks of all 35 batches had acceptable resolutions, with mangiferin exhibiting a good linear relationship in the range of 5.3291-133.2276 mg/L, and isomangiferin exhibiting a similar linear relationship in the range of 4.1847-104.6170 mg/L. The average recovery of mangiferin was 101.7%-105.6%, with a relative standard deviation (RSD) of 0.63%-1.43%, while that of isomangiferin was 103.4%-105.5%, with an RSD of 0.60%-1.18%. Importantly, all RSD values were less than 1.43%, thereby indicating that our method meets the requirements of the Chinese Pharmacopoeia (2020 Edition). Among the 35 batches of samples, the contents of mangiferin and isomangiferin were higher in the 2020 and 2021 samples, and the content fluctuation range was smaller. Overall, the development of an accurate and reliable quality control method for Fufang Jinqiancao granules, and a reasonable and effective quality evaluation of Fufang Jinqiancao granule samples from different years was realized. We therefore expect that this study will provide a reference for establishing a more systematic and comprehensive quality control system.

GEM-PA-Based Subunit Vaccines of Crimean Congo Hemorrhagic Fever Induces Systemic Immune Responses in Mice.

The Crimean Congo Hemorrhagic Fever Virus (CCHFV) is a tick-borne bunyavirus of the Narovirus genus, which is the causative agent of Crimean Congo Hemorrhagic Fever (CCHF). CCHF is endemic in Africa, the Middle East, Eastern Europe and Asia, with a high case-fatality rate of up to 50% in humans. Currently, there are no approved vaccines or effective therapies available for CCHF. The GEM-PA is a safe, versatile and effective carrier system, which offers a cost-efficient, high-throughput platform for recovery and purification of subunit proteins for vaccines. In the present study, based on a GEM-PA surface display system, a GEM-PA based vaccine expressing three subunit vaccine candidates (G-GP, including G-eGN, G-eGC and G-NAb) of CCHFV was developed, displaying the ectodomains of the structural glycoproteins eGN, eGC and NAb, respectively. According to the immunological assays including indirect-ELISA, a micro-neutralization test of pseudo-virus and ELISpot, 5 µg GPBLP3 combined with Montanide ISA 201VG plus Poly (I:C) adjuvant (A-G-GP-5 µg) elicited GP-specific humoral and cellular immunity in BALB/c mice after three vaccinations via subcutaneous injection (s.c.). The consistent data between IgG subtype and cytokine detection, ELISpot and cytokine detection indicated balanced Th1 and Th2 responses, of which G-eGN vaccines could elicit a stronger T-cell response post-vaccination, respectively. Moreover, all three vaccine candidates elicited high TNF-α, IL-6, and IL-10 cytokine levels in the supernatant of stimulated splenocytes in vitro. However, the neutralizing antibody (nAb) was only detected in A-G-eGC and A-G-eGC vaccination groups with the highest neutralizing titer of 128, suggesting that G-eGC could elicit a stronger humoral immune response. In conclusion, the GEM-PA surface display system could provide an efficient and convenient purification method for CCHFV subunit antigens, and the G-GP subunit vaccine candidates will be promising against CCHFV infections with excellent immunogenicity.

A Visual Assay of a Loop-Mediated Isothermal Amplification Based Vertical Immunoassay for SARS-CoV-2 RNA Detection.

SARS-CoV-2 is a novel coronavirus that has caused a global pandemic. To date, 504,907,616 people have been infected and developed coronavirus disease 2019 (COVID-19). A rapid and simple diagnostic method is needed to control this pandemic. In this study, a visual nucleic acid detection method combining reverse transcription loop-mediated isothermal amplification and a vertical flow visualization strip (RT-LAMP-VF) was successfully established and could detect 20 copies/µl of SARS-CoV-2 RNA transcript within 50 min at 61°C. This assay had no cross-reactivity with a variety of coronaviruses, including human coronavirus OC43, 229E, HKU1, NL63, severe acute respiratory syndrome-related coronavirus (SARSr-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and bat coronavirus HKU4, exhibiting very high levels of diagnostic sensitivity and specificity. Most strikingly, this method can be used for detecting multiple SARS-CoV-2 variants, including the Wuhan-Hu-1 strain, Delta, and Omicron variants. Compared with the RT-qPCR method recommended by the World Health Organization (WHO), RT-LAMP-VF does not require special equipment and is easy to perform. As a result, it is more suitable for rapid screening of suspected SARS-CoV-2 samples in the field and local laboratories.

Blending Technology Based on HPLC Fingerprint and Nonlinear Programming to Control the Quality of Ginkgo Leaves.

The breadth and depth of traditional Chinese medicine (TCM) applications have been expanding in recent years, yet the problem of quality control has arisen in the application process. It is essential to design an algorithm to provide blending ratios that ensure a high overall product similarity to the target with controlled deviations in individual ingredient content. We developed a new blending algorithm and scheme by comparing different samples of ginkgo leaves. High-consistency samples were used to establish the blending target, and qualified samples were used for blending. Principal component analysis (PCA) was used as the sample screening method. A nonlinear programming algorithm was applied to calculate the blending ratio under different blending constraints. In one set of calculation experiments, the result was blended by the same samples under different conditions. Its relative deviation coefficients (RDCs) were controlled within ±10%. In another set of calculations, the RDCs of more component blending by different samples were controlled within ±20%. Finally, the near-critical calculation ratio was used for the actual experiments. The experimental results met the initial setting requirements. The results show that our algorithm can flexibly control the content of TCMs. The quality control of the production process of TCMs was achieved by improving the content stability of raw materials using blending. The algorithm provides a groundbreaking idea for quality control of TCMs.