Grafted human-induced pluripotent stem cells-derived oligodendrocyte progenitor cells combined with human umbilical vein endothelial cells contribute to functional recovery following spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is a devastating disease that causes extensive damage to oligodendrocytes and neurons leading to demyelination and axonal degeneration. In this study, we co-transplanted cell grafts containing oligodendrocyte progenitor cells (OPCs) derived from human-induced pluripotent stem cells (iPSCs) combined with human umbilical vein endothelial cells (HUVECs), which were reported to promote OPCs survival and migration, into rat contusion models to promote functional recovery after SCI. METHODS: OPCs were derived from iPSCs and identified by immunofluorescence at different time points. Functional assays in vitro were performed to evaluate the effect of HUVECs on the proliferation, migration, and survival of OPCs by co-culture and migration assay, as well as on the neuronal axonal growth. A combination of OPCs and HUVECs was transplanted into the rat contusive model. Upon 8 weeks, immunofluorescence staining was performed to test the safety of transplanted cells and to observe the neuronal repairment, myelination, and neural circuit reconstruction at the injured area; also, the functional recovery was assessed by Basso, Beattie, and Bresnahan open-field scale, Ladder climb, SEP, and MEP. Furthermore, the effect of HUVECs on grafts was also determined in vivo. RESULTS: Data showed that HUVECs promote the proliferation, migration, and survival of OPCs both in vitro and in vivo. Furthermore, 8 weeks upon engraftment, the rats with OPCs and HUVECs co-transplantation noticeably facilitated remyelination, enhanced functional connection between the grafts and the host and promoted functional recovery. In addition, compared with the OPCs-alone transplantation, the co-transplantation generated more sensory neurons at the lesion border and significantly improved the sensory functional recovery. CONCLUSIONS: Our study demonstrates that transplantation of OPCs combined with HUVECs significantly enhances both motor and sensory functional recovery after SCI. No significance was observed between OPCs combined with HUVECs group and OPCs-alone group in motor function recovery, while the sensory function recovery was significantly promoted in OPCs combined with HUVECs groups compared with the other two groups. These findings provide novel insights into the field of SCI research.

A novel photosensitizer DTPP-mediated photodynamic therapy induces oxidative stress and apoptosis through mitochondrial pathways in LA795 cells.

OBJECTIVE: Investigation of the effects of 5-5- (4-N, N-diacetoxylphenyl)-10,15,20- tetraphenylporphyrin (DTPP)-mediated photodynamic therapy (PDT) on oxidative stress and mitochondrial apoptosis in LA795 lung cancer cells. METHODS: Proteomics was used to identify differentially expressed proteins after PDT treatment. The apoptosis rate was determined by flow cytometry. Morphologic observation of apoptosis, reactive oxygen species (ROS) levels, antioxidant indices, nitric oxide (NO) content, mitochondrial membrane potential (MMP), and Caspase- 9 and Caspase-3 were determined by assays; apoptosis-related protein levels of Cytochrome (Cyto) c, Bcl- 2, Bax were determined by Western blot. RESULTS: Typical apoptosis morphology of LA795 cells was observed after PDT. The cells were mainly in the apoptosis death pathway with high cell apoptosis rates. The proteomics study observed the apoptosis-associated proteins, oxidative stress proteins, antioxidant proteins, the cytoskeletal protein and mitochondrial dysfunction in LA 795 cells. Additional results indicated that PDT could increase levels of ROS, NO; decrease glutathione (GSH) content and MMP; upregulated Bax, Cyto c, and Caspase-3 protein expression, inhibited Bcl-2 protein expression, and further induced cell apoptosis. The effect of DTPP-PDT on lung cancer was: first, mitochondrial Cyto c is released into the cytoplasm, then Caspase- 9 / Caspase-3 was activated, Bcl-2 decreased/Bax increased, initiating cell apoptosis. CONCLUSION: DTPP-PDT could induce oxidative stress and apoptosis via mitochondrial pathways in LA795 cells.

Three-Dimensional Printing of Large Objects with High Resolution by Dynamic Projection Scanning Lithography.

Due to the development of printing materials, light-cured 3D printing is playing an increasingly important role in industrial and consumer markets for prototype manufacturing and conceptual design due to its advantages in high-precision and high-surface finish. Despite its widespread use, it is still difficult to achieve the 3D printing requirements of large volume, high resolution, and high speed. Currently, traditional light-cured 3D printing technologies based on stereolithography, such as regular DLP and SLA, can no longer meet the requirements of the processing size and processing rate. This paper introduces a dynamic projection of 3D printing technology utilizing a digital micro-mirror device (DMD). By projecting the ultraviolet light pattern in the form of "animation", the printing resin is continuously cured in the exposure process to form the required three-dimensional structure. To print large-size objects, the three-dimensional model is sliced into high-resolution sectional images, and each layer of the sectional image is further divided into sub-regional images. These images are dynamically exposed to the light-curing material and are synchronized with the scanning motion of the projection lens to form a static exposure pattern in the construction area. Combined with the digital super-resolution, this system can achieve the layering and fine printing of large-size objects up to 400 × 400 × 200 mm, with a minimum feature size of 45 µm. This technology can achieve large-size, high-precision structural printing in industrial fields such as automobiles and aviation, promoting structural design, performance verification, product pre-production, and final part processing. Its printing speed and material bending characteristics are superior to existing DLP light-curing 3D printing methods.

Occult Hepatitis B Virus Infection and Liver Fibrosis in Chinese Patients.

BACKGROUND: The impact of hepatitis B surface antigen (HBsAg)-negative/hepatitis B virus (HBV) DNA-positive occult HBV infection (OBI) on the severity of liver fibrosis remains unclear. METHODS: A total of 1772 patients negative for HBsAg but positive for antibody to hepatitis B core antigen (HBcAg), stratified by the presence or absence of OBI, were selected for long-term carriage leading to elevation of ≥2 of 4 liver fibrosis indexes-hyaluronic acid (HA), laminin, type III procollagen peptide (PCIII), and type IV collagen (CIV)-at testing in a Chinese hospital. Patients were tested for serum viral load, HBV markers, and histopathological changes in liver biopsy specimens. RESULTS: OBI was identified in 148 patients with liver fibrosis (8.4%), who had significantly higher levels of HA, laminin, PCIII, and CIV than 1624 fibrotic patients without OBI (P < .05). In 36 patients with OBI who underwent liver biopsy, significant correlations were observed between OBI viral load and serum HA levels (P = .01), PCIII levels (P = .01), and pathological histological activity index (HAI) scores (P < .001), respectively; HAI scores and PCIII levels (P = .04); HBcAg immunohistochemical scores and HA levels (P < .001); and HBcAg immunohistochemical scores and PCIII levels (P = .03). Positive fluorescent in situ hybridization results were significantly more frequent in patients with OBIs (80.6% vs 37.5% in those without OBIs). Among patients with OBIs, HBcAg was detected in the liver tissue in 52.8% and HBsAg in 5.6%. CONCLUSIONS: OBI status appears to be associated with liver fibrosis severity.

Early Phase of Specific Cellular Immune Status Associates with HCV Infection Outcomes in Marmosets.

The major mechanism for determination of HCV infection outcomes has not been fully described, particularly in the early phase of the "window-period" of infection. Based on two groups of marmosets infected with HCV-CE1E2p7/GBV-B chimeric virus (HCV chimera) or GBV-B, the immune mechanism correlating with the different outcomes of virus infections was explored in this study. HCV chimera containing the entire HCV core and envelope proteins (CE1E2p7) and GBV-B RNA were intrahepatically injected into four marmosets in each group, respectively. Blood samples were taken from individual animals in an interval of 2 weeks. Viral load and specific T cell responses were detected in two groups of HCV chimera- and GBV-B-infected marmosets. HCV chimera-infected marmosets appeared to have a virally persistent infection over 6 months post inoculation of the virus. Of these, the specific IFN-γ-secretion T cell response slowly developed over 13 to 19 weeks and was maintained at a relatively low level with 40-70 SFC/106 PBMCs, while the specific Treg cell response was rapidly activated over 3 weeks and was maintained at a high level around 5% among lymphocytes. In contrast, GBV-B-infected marmosets presented spontaneous viral clearance within 6 months; the specific IFN-γ-secretion T cell response was quickly established over 5 to 7 weeks and was maintained at a high level with 50-130 SFC/106 PBMCs, while the specific Treg cell response was inactivated and maintained at a baseline below 3% among lymphocytes. In conclusion, the HCV structural proteins inducing immune suppression in the early phase of HCV infection contributed to the viral persistence, of which the activation of Treg cells might play an important role in the inhibition of an effective T cell antiviral response.

A Helper Antibody-Based Competitive Fluorescence Immunochromatographic Assay for Quantitative Detection of Florfenicol in Poultry Eggs.

BACKGROUND: Florfenicol (FF) is a chloramphenicol analogue used in animals, and florfenicol amine (FFA) is the main metabolite of FF. However, their residues in agricultural products are harmful to human health. A highly specific and sensitive assay for FF/FFA detection needs to be developed since the traditional detection methods are low in sensitivity. OBJECTIVE: In this study, a new method for rapid quantification of FF/FFA in poultry eggs by helper antibody-based fluorescent immunochromatographic assay (HAFIA) was established. METHODS: Triple antibodies including a primary monoclonal antibody (mAb) specific to the targets FF and FFA, a secondary polyclonal antibody (pAb) labeled with europium nanoparticles (EuNPs), and a helper monoclonal antibody (hAb), reacting with pAb but not with the mAb or the target antigen, are designed, which can form structural aggregation complexes in microwells with a single step of reactions. By loading the reaction sample solution, the triple-antibodies (mAb-pAb-hAb)-EuNPs complexes migrate to the test (T) line on the nitrocellulose membrane of testing strip and are competitively captured by the immobilized FF-bovine serum album (BSA) conjugates on the membrane and the FF/FFA targets in the sample solution. RESULTS: Fluorescence on the T line is read by a portable fluorescent strip reader in 10 min, and the result is given as the ratio of fluorescent intensities on the T and control (C) lines. This new fluorescent testing strip, with amplified signal from the triple-antibody complex, has 50-fold higher sensitivity than conventional colloidal gold-lateral flow immunoassays (CG-LFIAs), and can detect as low as 0.01 ng/mL FF and 0.1 ng/mL FFA targets from egg samples. CONCLUSION: The developed competitive fluorescent immunochromatography method based on auxiliary antibodies has the advantages of high sensitivity and specificity for the rapid and quantitative detection of FF/FFA in poultry eggs. HIGHLIGHTS: Newly designed helper antibody and portable device were applied to quantitative detection. HAFIA tests egg samples and results can be obtained in 10 minutes. HAFIA has the advantages of being more convenient, faster and does not require professional laboratory personnel.

Smartphone-Based High-Throughput Fiber-Integrated Immunosensing System for Point-of-Care Testing of the SARS-CoV-2 Nucleocapsid Protein.

To control the coronavirus disease 2019 (COVID-19) pandemic, there is an urgent need for simple, rapid, and reliable detection methods to identify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, especially in community hospitals or clinical centers. The SARS-CoV-2 nucleocapsid protein (NP) is an important index for diagnosis of COVID-19. Here, we proposed a smartphone-based high-throughput fiber-integrated immunosensing system (HFIS) for detecting the SARS-CoV-2 NP in serum samples within 45 min. For the testing of NP standards, the linear detection range was 7.8-1000 pg/mL, the limit of detection was 7.5 pg/mL, and the cut-off value was 8.923 pg/mL. Twenty-five serum samples from clinically diagnosed COVID-19 patients and 100 negative control samples from healthy blood donors were tested for SARS-CoV-2 NP by HFIS, and the obtained results were compared with those of ELISA and Simple Western analysis. The results showed that the HFIS sensitivity and specificity were 72% [95% confidence interval (CI): 52.42-85.72%] and 100% (95% CI: 96.11-100%), respectively, which significantly correlated with those from the commercial ELISA kit and Simple Western analysis. This portable high-throughput HFIS assay could be an alternative test for detecting SARS-CoV-2 NP in blood samples on site.

SETD4-mediated KU70 methylation suppresses apoptosis.

The mammalian KU70 is a pleiotropic protein functioning in DNA repair and cytoplasmic suppression of apoptosis. We report a regulatory mechanism by which KU70's cytoplasmic function is enabled due to a methylation at K570 of KU70 by SET-domain-containing protein 4 (SETD4). While SETD4 silencing reduces the level of methylated KU70, over-expression of SETD4 enhances methylation of KU70. Mutations of Y272 and Y284 of SETD4 abrogate methylation of KU70. Although SETD4 is predominantly a nuclear protein, the methylated KU70 is enriched in the cytoplasm. SETD4 knockdown enhances staurosporine (STS)-induced apoptosis and cell killing. Over-expression of the wild-type (WT) SETD4, but not the SETD4-Y272/Y284F mutant, suppresses STS-induced apoptosis. The KU70-K570R (mouse Ku70-K568R) mutation dampens the anti-apoptosis activity of KU70. Our study identifies KU70 as a non-histone substrate of SETD4, discovers a post-translational modification of KU70, and uncovers a role for SETD4 and KU70-K570 methylation in the suppression of apoptosis.

Development of a smartphone-based quantum dot lateral flow immunoassay strip for ultrasensitive detection of anti-SARS-CoV-2 IgG and neutralizing antibodies.

BACKGROUND: As several vaccines for SARS-CoV-2 have been developed, a large proportion of individuals have been vaccinated worldwide so far. The rapid and accurate immunoassays are urgently needed for detecting the specific virus-neutralizing antibody (NAb), which reflect the protective effect of the vaccines among different populations. METHODS: In this study, we designed a quantum dot lateral flow immunoassay strip (QD-LFIA) for smartphones for the detection of specific IgG or neutralizing antibodies in SARS-CoV-2 in human serum or whole blood samples. The recombinant receptor binding domain of the SARS-CoV-2 spike protein was used as the antigen to combine with NAb or angiotensin-converting enzyme 2. RESULTS: Among 81 patients who recovered from COVID-19 who were diagnosed using the nucleic acid test initially, 98.8% (80/81) were positive for IgG and 88.9% (72/81) were positive for NAb by QD-LFIA. Among 64 individuals inoculated with inactivated vaccines and six subunit vaccines, 90% (63/70) were positive for IgG and 82.9% (58/70) were positive for NAb by QD-LFIA, whereas no cross-reaction was found in 150 healthy blood donors, two patients with influenza B, and three patients with common cold. CONCLUSION: The established platform could achieve a rapid and accurate detection of NAb specific to SARS-CoV-2, which could be used for detecting the protective effect of the vaccines in areas of world that currently affected by the pandemic.

Hepatitis B Virus-Specific Cellular Immunity Contributes to the Outcome of Occult Hepatitis B Virus Infection.

There is little known of immunologic factors leading to the occurrence of occult HBV infection (OBI). Specific cellular immune response to hepatitis B virus (HBV) core/pol peptides was compared between blood donor populations, including 37 OBIs, 53 chronic HBV infections (CHB), 47 resolved infections, and 56 non-infected controls, respectively. The rate of CD4+/CD8+ T cell proliferation in OBI or CHB carriers was higher than in HBV resolved and non-infected individuals (P < 0.05). The intensity of IFN-γ-secretion T-cell response of OBI carriers was highest, followed by CHB and resolved infections, and non-infected individuals (P < 0.05). The frequency of intracellular IFN-γ and IL-17A CD4+/CD8+ and IL-21 CD4+ T-cell responses was significantly higher in resolved infections than in OBI or CHB carriers (P < 0.05), while the level of extracellular IL-17A of peripheral blood mononuclear cells (PBMCs) was higher in OBI and CHB carriers than in resolved infections (P < 0.01). The frequency of intracellular IL-10 CD4+ T-cell response in CHB, OBI, and resolved infections was higher than in HBV non-infected individuals (P < 0.01). Intracellular IL-10 CD8+ T cell and extracellular IL-10 T-cell responses were higher in CHB than in OBI (P = 0.012) or HBV resolved infections (P < 0.01). In conclusion, the higher level of effective T-cell response with IFN-γ, IL-17A, and IL-21 contributes to resolved infection outcome, while higher levels of suppressive T-cell response with IL-10 result in HBV chronicity. OBI is an intermediary status between HBV resolved and chronic infections, in which IL-21 effector and IL-10 suppressor T-cell responses play an important role in directing the outcome of HBV infection.

Motor neuron replacement therapy for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a motor neuron degenerative disease that is also known as Lou Gehrig's disease in the United States, Charcot's disease in France, and motor neuron disease in the UK. The loss of motor neurons causes muscle wasting, paralysis, and eventually death, which is commonly related to respiratory failure, within 3-5 years after onset of the disease. Although there are a limited number of drugs approved for amyotrophic lateral sclerosis, they have had little success at treating the associated symptoms, and they cannot reverse the course of motor neuron degeneration. Thus, there is still a lack of effective treatment for this debilitating neurodegenerative disorder. Stem cell therapy for amyotrophic lateral sclerosis is a very attractive strategy for both basic and clinical researchers, particularly as transplanted stem cells and stem cell-derived neural progenitor/precursor cells can protect endogenous motor neurons and directly replace the lost or dying motor neurons. Stem cell therapies may also be able to re-establish the motor control of voluntary muscles. Here, we review the recent progress in the use of neural stem cells and neural progenitor cells for the treatment of amyotrophic lateral sclerosis. We focus on MN progenitor cells derived from fetal central nervous system tissue, embryonic stem cells, and induced pluripotent stem cells. In our recent studies, we found that transplanted human induced pluripotent stem cell-derived motor neuron progenitors survive well, differentiate into motor neurons, and extend axons into the host white matter, not only in the rostrocaudal direction, but also along motor axon tracts towards the ventral roots in the immunodeficient rat spinal cord. Furthermore, the significant motor axonal extension after neural progenitor cell transplantation in amyotrophic lateral sclerosis models demonstrates that motor neuron replacement therapy could be a promising therapeutic strategy for amyotrophic lateral sclerosis, particularly as a variety of stem cell derivatives, including induced pluripotent stem cells, are being considered for clinical trials for various diseases.

Characterization of Human-Induced Neural Stem Cells and Derivatives following Transplantation into the Central Nervous System of a Nonhuman Primate and Rats.

Neural stem cells (NSCs) and derivatives are potential cellular sources to treat neurological diseases. In the current study, we reprogrammed human peripheral blood mononuclear cells into induced NSCs (iNSCs) and inserted GFP gene into the AAVS1 site for graft tracing. Targeted integration of GFP does not affect the proliferation and differentiation capacity of iNSCs. iNSC-GFP can be further differentiated into dopaminergic precursors (DAPs) and motor neuron precursors (MNPs), respectively. iNSCs were engrafted into the motor cortex and iNSC-DAPs into the striatum and substantia nigra (SN) of a nonhuman primate, respectively. The surviving iNSCs could respond to the microenvironment of the cortex and spontaneously differentiate into mature neurons that extended neurites. iNSC-DAPs survived well and matured into DA neurons following transplantation into the striatum and SN. iNSC-MNPs could also survive and turn into motor neurons after being engrafted into the spinal cord of rats. The results suggest that iNSCs and derivatives have a potential to be used for the treatment of neurological diseases.

Learning Privacy-Preserving Student Networks via Discriminative-Generative Distillation.

While deep models have proved successful in learning rich knowledge from massive well-annotated data, they may pose a privacy leakage risk in practical deployment. It is necessary to find an effective trade-off between high utility and strong privacy. In this work, we propose a discriminative-generative distillation approach to learn privacy-preserving deep models. Our key idea is taking models as bridge to distill knowledge from private data and then transfer it to learn a student network via two streams. First, discriminative stream trains a baseline classifier on private data and an ensemble of teachers on multiple disjoint private subsets, respectively. Then, generative stream takes the classifier as a fixed discriminator and trains a generator in a data-free manner. After that, the generator is used to generate massive synthetic data which are further applied to train a variational autoencoder (VAE). Among these synthetic data, a few of them are fed into the teacher ensemble to query labels via differentially private aggregation, while most of them are embedded to the trained VAE for reconstructing synthetic data. Finally, a semi-supervised student learning is performed to simultaneously handle two tasks: knowledge transfer from the teachers with distillation on few privately labeled synthetic data, and knowledge enhancement with tangent-normal adversarial regularization on many triples of reconstructed synthetic data. In this way, our approach can control query cost over private data and mitigate accuracy degradation in a unified manner, leading to a privacy-preserving student model. Extensive experiments and analysis clearly show the effectiveness of the proposed approach.

A nanoenzyme linked immunochromatographic sensor for rapid and quantitative detection of SARS-CoV-2 nucleocapsid protein in human blood.

The establishment of a simple, low-cost, high-sensitive and rapid immunoassay for detecting SARS-CoV-2 antigen in human blood is an effective mean of discovering early SARS-CoV-2 infection and controlling the pandemic of COVID-19. Herein, a smartphone based nanozyme linked immunochromatographic sensor (NLICS) for the detection of SARS-CoV-2 nucleocapsid protein (NP) has been developed on demand. The system is integrated by disposable immunochromatography assay (ICA) and optical sensor devices. Immunoreaction and enzyme-catalyzed substrate color reaction were carried out on the chromatographic strip in a device, of which the light signal was read by a photometer through a biosensor channel, and the data was synchronously transmitted via the Bluetooth to the app in-stored smartphone for reporting the result. With a limit of detection (LOD) of 0.026 ng/mL NP, NLICS had the linear detection range (LDR) between 0.05 and 1.6 ng/mL NP, which was more sensitive than conventional ICA. NLICS took 25 min for reporting results. For detection of NP antigen in clinical serum samples from 21 COVID-19 patients and 80 healthy blood donor controls, NLICS and commercial enzyme linked immunosorbent assay (ELISA) had 76.2% or 47.6% positivity, and 100% specificity, respectively (P = 0.057), while a good correlation coefficient (r = 0.99) for quantification of NP between two assays was obtained. In conclusion, the NLICS was a rapid, simple, cheap, sensitive and specific immunochromatographic sensing assay for early diagnosis of SARS-CoV-2 infection.

A Self-Biomineralized Novel Adenovirus Vectored COVID-19 Vaccine for Boosting Immunization of Mice.

SARS-CoV-2 has caused more than 3.8 million deaths worldwide, and several types of COVID-19 vaccines are urgently approved for use, including adenovirus vectored vaccines. However, the thermal instability and pre-existing immunity have limited its wide applications. To circumvent these obstacles, we constructed a self-biomineralized adenovirus vectored COVID-19 vaccine (Sad23L-nCoV-S-CaP) by generating a calcium phosphate mineral exterior (CaP) based on Sad23L vector carrying the full-length gene of SARS-CoV-2 spike protein (S) under physiological condition. This Sad23L-nCoV-S-CaP vaccine was examined for its characteristics of structure, thermostability, immunogenicity and avoiding the problem of preexisting immunity. In thermostability test, Sad23L-nCoV-S-CaP could be stored at 4 °C for over 45 days, 26 °C for more than 8 days and 37 °C for approximately 2 days. Furthermore, Sad23L-nCoV-S-CaP induced higher level of S-specific antibody and T cell responses, and was not affected by the pre-existing anti-Sad23L immunity, suggesting it could be used as boosting immunization on Sad23L-nCoV-S priming vaccination. The boosting with Sad23L-nCoV-S-CaP vaccine induced high titers of 105.01 anti-S1, 104.77 anti-S2 binding antibody, 103.04 pseudovirus neutralizing antibody (IC50), and robust T-cell response of IFN-γ (1466.16 SFCs/106 cells) to S peptides, respectively. In summary, the self-biomineralization of the COVID-19 vaccine Sad23L-nCoV-S-CaP improved vaccine efficacy, which could be used in prime-boost regimen for prevention of SARS-CoV-2 infection in humans.

Development of a Smartphone-Based Nanozyme-Linked Immunosorbent Assay for Quantitative Detection of SARS-CoV-2 Nucleocapsid Phosphoprotein in Blood.

Since December 2019, a novel coronavirus (SARS-CoV-2) has resulted in a global pandemic of coronavirus disease (COVID-19). Although viral nucleic acid test (NAT) has been applied predominantly to detect SARS-CoV-2 RNA for confirmation diagnosis of COVID-19, an urgent need for alternative, rapid, and sensitive immunoassays is required for primary screening of virus. In this study, we developed a smartphone-based nanozyme-linked immunosorbent assay (SP-NLISA) for detecting the specific nucleocapsid phosphoprotein (NP) of SARS-CoV-2 in 37 serum samples from 20 COVID-19 patients who were diagnosed by NAT previously. By using SP-NLISA, 28/37 (75.7%) serum samples were detected for NP antigens and no cross-reactivity with blood donors' control samples collected from different areas of China. In a control assay using the conventional enzyme-linked immunosorbent assay (ELISA), only 7/37 (18.91%) serum samples were detected for NP antigens and no cross-reactivity with control samples. SP-NLISA could be used for rapid detection of SARS-CoV-2 NP antigen in primary screening of SARS-CoV-2 infected individuals.

Track-etched membrane microplate and smartphone immunosensing for SARS-CoV-2 neutralizing antibody.

The level of neutralizing antibody (NAb) to SARS-CoV-2 could be used to evaluate the acquired protective immunity of COVID-19 patients or vaccinees. Here we develop a track-etched microporous membrane filtration microplate (TEM) and optical fibers transmitted immunosensing smartphone platform (TEMFIS) based surrogate virus neutralization test (TEMFIS-sVNT) for rapid one-step testing of NAb to SARS-CoV-2. Coefficient variation (CV) of intra-assay and inter-assay precisions of TEMFIS-sVNT varied below 9% or 14%, respectively. By agreement with pseudovirus neutralization test (pVNT) and ELISA-sVNT for testing of serum samples from 41 COVID-19 patients, 50 COVID-19 vaccinees and 320 healthy blood donors (P = 0.895), TEMFIS-sVNT detected the NAb positivity (sensitivity) in 92.68% COVID-19 patients and 76% vaccinees, but the NAb negativity (specificity) in 100% blood donors. In conclusion, TEMFIS-sVNT can be used for quantitatively point-of-care testing of neutralizing antibody to SARS-CoV-2 in blood samples from COVID-19 patients and vaccinees.

Prime-boost vaccination of mice and rhesus macaques with two novel adenovirus vectored COVID-19 vaccine candidates.

ABSTRACTCOVID-19 vaccines are being developed urgently worldwide. Here, we constructed two adenovirus vectored COVID-19 vaccine candidates of Sad23L-nCoV-S and Ad49L-nCoV-S carrying the full-length gene of SARS-CoV-2 spike protein. The immunogenicity of two vaccines was individually evaluated in mice. Specific immune responses were observed by priming in a dose-dependent manner, and stronger responses were obtained by boosting. Furthermore, five rhesus macaques were primed with 5 × 109 PFU Sad23L-nCoV-S, followed by boosting with 5 × 109 PFU Ad49L-nCoV-S at 4-week interval. Both mice and macaques well tolerated the vaccine inoculations without detectable clinical or pathologic changes. In macaques, prime-boost regimen induced high titers of 103.16 anti-S, 102.75 anti-RBD binding antibody and 102.38 pseudovirus neutralizing antibody (pNAb) at 2 months, while pNAb decreased gradually to 101.45 at 7 months post-priming. Robust T-cell response of IFN-γ (712.6 SFCs/106 cells), IL-2 (334 SFCs/106 cells) and intracellular IFN-γ in CD4+/CD8+ T cell (0.39%/0.55%) to S peptides were detected in vaccinated macaques. It was concluded that prime-boost immunization with Sad23L-nCoV-S and Ad49L-nCoV-S can safely elicit strong immunity in animals in preparation of clinical phase 1/2 trials.

A time-resolved fluorescence lateral flow immunoassay for rapid and quantitative serodiagnosis of Brucella infection in humans.

Brucellosis is a worldwide infectious zoonotic disease, posing severe threats to human health and social-economic development. By comparing with time-consuming, low sensitive and non-quantitative conventional serological methods, herein, protein G (prG) coupled with europium nanospheres (EuNPs) (detection probe) and highly purified Brucella lipopolysaccharide (LPS) (capture antigen) were used to develop a novel time-resolved fluorescence lateral flow immunoassay (TF-LFIA) for detecting anti-Brucella IgG antibody in human plasmas. The entire testing took 15 min. With a satisfactory purity, the purified LPS weakly cross-reacted with Y. enterocolitica O9 diagnostic antibody; however, none reacted with sera from patients with other Gram-negative bacterial infections. Following coefficient of determination (R2 = 0.9961), 0.3 IU/mL was reported as the limit of detection (LOD), much lower than those of Serological Agglutination Test (SAT), Rose-Bengal Plate Agglutination Test (RBPT) and colloidal gold LFIA (CG-LFIA). Intra-day and inter-day precisions (CV, coefficient variation) of TF-LFIA varied less than 8% or 12 %, while intra-day and inter-day accuracies were 94-106 % or 93-107 %, respectively. The correlation coefficient (R2) of TF-LFIA measurement to the different concentrations of spiked Brucella antibody was 0.9967, suggesting TF-LFIA had high reliability and reproducibility. TF-LFIA was demonstrated for 100 % specificity, 98.57 % sensitivity and 99.63 % accuracy in detection of Brucella antibody from clinical samples, respectively, significantly higher compared to SAT and RBPT. In conclusion, the established TF-LFIA is a simple, rapid and quantitative immunoassay for early diagnosis or epidemiological surveillance of Brucella infection in humans.

Role of core protein mutations in the development of occult HBV infection.

BACKGROUND & AIMS: Occult HBV infection (OBI) is associated with transfusion-transmitted HBV infection and hepatocellular carcinoma. Studies on OBI genesis have concentrated on mutations in the S region and the regulatory elements. Herein, we aimed to determine the role of mutations in the core region on OBIs. METHODS: An OBI strain (SZA) carrying 9 amino acid (aa) substitutions in the core protein/capsid (Cp) was selected by sequence alignment and Western blot analysis from 26 genotype B OBI samples to extensively explore the impact of Cp mutations on viral antigen production in vitro and in vivo. RESULTS: A large panel of 30 Cp replicons were generated by a replication-competent pHBV1.3 carrying SZA or wild-type (WT) Cp in a 1.3-fold over-length of HBV genome, in which the various Cp mutants were individually introduced by repairing site mutations of SZA-Cp or creating site mutations of WT-Cp by site-directed mutagenesis. The expression of HBcAg, HBeAg, and HBsAg and viral RNA was quantified from individual SZA and WT Cp mutant replicons in transfected Huh7 cells or infected mice, respectively. An analysis of the effect of Cp mutants on intracellular or extracellular viral protein production indicated that the W62R mutation in Cp had a critical impact on the reduction of HBcAg and HBeAg production during HBV replication, whereas P50H and/or S74G mutations played a limited role in influencing viral protein production invivo. CONCLUSIONS: W62R and its combination mutations in HBV Cp might massively affect HBcAg and HBeAg production during viral replication, which, in turn, might contribute to the occurrence of OBI. LAY SUMMARY: Occult hepatitis B virus infections (OBIs) have been found to be associated with amino acid mutations in the S region of the HBV, but the role of mutations in the core protein (Cp) remains unclear. In this study, an OBI strain (SZA) carrying 9 amino acid substitutions in Cp has been examined comprehensively in vitro and in vivo. The W62R mutation in Cp majorly reduces HBcAg and HBeAg production during HBV replication, potentially contributing to the occurrence of OBI.