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.

Vaccination of cats with Sad23L-nCoV-S vaccine candidate against major variants of SARS-CoV-2.

Cats are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and risk transmitting viruses to naive cats or humans. Here, based on our novel adenovirus-vectored COVID-19 vaccine, the immunogenicity of Sad23L-nCoV-S vaccine was evaluated in cats by prime-boost vaccinations. Five cats were primed with a dose of 108 plaque-forming units (PFUs) Sad23L-nCoV-S vaccine and then boosted with an equal dose of same vaccine at a 4-week interval. Cat serum neutralizing antibody (NAb) titers (the sample dilution at which 50% inhibitory concentration [IC50]) were measured as IC50 15,849 to wild-type strain, IC50 6,591 to Alpha, IC50 2,315 to Beta, IC50 2,744 to Gamma, IC50 1,848 to Delta, and IC50 318 to Omicron variants of pseudotyped SARS-CoV-2 viruses at week 6 post-prime vaccination. All NAb levels to these five variants were ≥IC50 49 from vaccinated cats at week 10, while 48.8% to Delta and 100% to Omicron variants were

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.

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.

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.

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.

Low prevalence of antibodies against SARS-CoV-2 among voluntary blood donors in Guangzhou, China.

Since the first case of COVID-19 reported in late December of 2019 in Wuhan, China, the SARS-CoV-2 virus has caused approximately 20 million infections and 732 thousand deaths around the world by 11 August 2020. Although the pathogen generally infects the respiratory system, whether it is present in the bloodstream and whether it poses a threat to the blood supply during the period of the outbreak is of serious public concern. In this study, we used enzyme-linked immunosorbent assay (ELISA) to screen total antibodies against SARS-CoV-2 in 2199 blood donors, who had donated blood at the Guangzhou Blood Center during the epidemic. The Ig-reactive samples were further characterized for IgA, IgG, and IgM subtypes by ELISA and viral nucleic acid by real-time polymerase chain reaction. Among the 2199 plasma samples, seven were reactive under total antibodies' screening. Further testing revealed that none of them had detectable viral nucleic acid or IgM antibody, but two samples contained IgA and IgG. The IgG antibody titers of both positive samples were 1:16 and 1:4, respectively. Our results indicated a low prevalence of past SARS-CoV-2 infection in our blood donors, as none of the tests were positive for viral nucleic acid and only 2 out of 2199 (0.09%) of samples were positive for IgG and IgA. There would be a limited necessity for the implementation of such testing in blood screening in a COVID-19 low-risk area.