Nanobodies against SARS-CoV-2 RBD from a Two-step Phage Screening of Universal and Focused Synthetic Libraries (preprint)

Coronavirus disease 2019 (COVID-19) is an evolving global pandemic, and nanobody (Nb) is recognized as a potential diagnostic and therapeutic tool for infectious disease. Here, we designed and synthesized a humanized and highly diverse phage Nbs library hsNb-U (Humanized synthetic Nbs Library - Universal). We expressed and purified the SARS-CoV-2 receptor-binding domain (RBD), and screened this univeral library against the RBD protein target. Then, the CDR1 and CDR2 sequences of five leads obtained from the hsNb-U phage panning were combined with randomly mutated CDR3 to construct a targeted (focused) phage display library, hsNb-RBD, for subsequent phage panning and screening. From the obtained sequences, we expressed 45 unique anti-RBD candidate Nbs. Among the selected Nbs, eight were found to be highly expressed, and five of these show high-affinity to RBD (EC50 less than 100nM). Finally, we found that Nb39 can compete with angiotensin converting enzyme 2 (ACE2) for binding to RBD. Overall, this two-step strategy of synthetic phage display libraries enables rapid selection of SARS-CoV-2 RBD nanobodies with potential therapeutic activity, and this two-step strategy can potentially be used for rapid discovery of Nbs against other targets.

A potent, broadly protective vaccine against SARS-CoV-2 variants of concern.

Since the first outbreak in December 2019, SARS-CoV-2 has been constantly evolving and five variants have been classified as Variant of Concern (VOC) by the World Health Organization (WHO). These VOCs were found to enhance transmission and/or decrease neutralization capabilities of monoclonal antibodies and vaccine-induced antibodies. Here, we successfully designed and produced a recombinant COVID-19 vaccine in CHO cells at a high yield. The vaccine antigen contains four hot spot substitutions, K417N, E484K, N501Y and D614G, based on a prefusion-stabilized spike trimer of SARS-CoV-2 (S-6P) and formulated with an Alum/CpG 7909 dual adjuvant system. Results of immunogenicity studies showed that the variant vaccine elicited robust cross-neutralizing antibody responses against SARS-CoV-2 prototype (Wuhan) strain and all 5 VOCs. It further, stimulated a TH1 (T Helper type 1) cytokine profile and substantial CD4+ T cell responses in BALB/c mice and rhesus macaques were recorded. Protective efficacy of the vaccine candidate was evaluated in hamster and rhesus macaque models of SARS-CoV-2. In Golden Syrian hamsters challenged with Beta or Delta strains, the vaccine candidate reduced the viral loads in nasal turbinates and lung tissues, accompanied by significant weight gain and relieved inflammation in the lungs. In rhesus macaque challenged with prototype SARS-CoV-2, the vaccine candidate decreased viral shedding in throat, anal, blood swabs over time, reduced viral loads of bronchus and lung tissue, and effectively relieved the lung pathological inflammatory response. Together, our data demonstrated the broadly neutralizing activity and efficacy of the variant vaccine against both prototype and current VOCs of SARS-CoV-2, justifying further clinical development.

Advances in nanoparticle‐based lateral flow immunoassay for point‐of‐care testing

As a representative technology for point‐of‐care testing (POCT), lateral flow immunoassay (LFIA) has been broadly used to detect analytes in many fields. However, its clinical application is severely limited by the unsatisfactory sensitivity, which makes it difficult to obtain accurate results when detecting biomarkers of trace levels, especially in complex matrices. Nanoparticles have been introduced into LFIA for years and become an indispensable part, acting not only as carriers that load and enrich biomolecules, such as antibodies and dyes, but also a miniature platform applied for creative design and construction of nanoprobes. Due to the unique properties at the nanoscale, including the mimetic enzyme activity, the characteristic plasma resonance spectrum and so on, nanomaterials exhibit great potential in the development of novel LFIA and high‐sensitivity detection.

Development of recombinant COVID-19 vaccine based on CHO-produced, prefusion spike trimer and alum/CpG adjuvants.

COVID-19 pandemic has severely impacted the public health and social economy worldwide. A safe, effective, and affordable vaccine against SARS-CoV-2 infections/diseases is urgently needed. We have been developing a recombinant vaccine based on a prefusion-stabilized spike trimer of SARS-CoV-2 and formulated with aluminium hydroxide and CpG 7909. The spike protein was expressed in Chinese hamster ovary (CHO) cells, purified, and prepared as a stable formulation with the dual adjuvant. Immunogenicity studies showed that candidate vaccines elicited robust neutralizing antibody responses and substantial CD4+ T cell responses in both mice and non-human primates. And vaccine-induced neutralizing antibodies persisted at high level for at least 6 months. Challenge studies demonstrated that candidate vaccine reduced the viral loads and inflammation in the lungs of SARS-CoV-2 infected golden Syrian hamsters significantly. In addition, the vaccine-induced antibodies showed cross-neutralization activity against B.1.1.7 and B.1.351 variants. These data suggest candidate vaccine is efficacious in preventing SARS-CoV-2 infections and associated pneumonia, thereby justifying ongoing phase I/II clinical studies in China (NCT04982068 and NCT04990544).

Impact of Home Confinement Due to COVID-19 Pandemic on Mental Health in Young Children: A Natural Experiment (preprint)

Background: The outbreak of coronavirus disease in 2019 (COVID-19) has had an enormous impact on people’s health, and profoundly changed our lives and society. However, the impact of pandemic-related home confinement on children’s life and their mental health remains unknown. We took advantage of a natural experiment due to the onset of COVID-19 and examined this issue in two consecutive cohorts. Methods: This study compared mental health, family environment and lifestyles of two representative cohorts of young children in Shanghai, China. Both prospective cohorts recruited children at entry to kindergarten. Surveys were conducted at both entry and graduation of kindergarten using the same assessment instruments. Children in the non-exposed cohort experienced a normal period of kindergarten study and graduated in June, 2019; those in the exposed cohort were under confinement for five months and graduated in June, 2020. Findings: Compared with the non-exposed cohort, the exposed cohort had a significant increased percentage of children classified as “at-risk for mental health problems”, with an adjusted absolute risk difference of 3·1% (95% CI, 1·9% to 4·4%) for total difficulties as measured by the Strength and Difficulty Questionnaire. The effect of pandemic related confinement on mental health was larger in children with lower parental education. The exposed cohort had longer media time, sleep duration and a less favorable home environment. Interpretation: The pandemic related confinement negatively affected children's mental health and home environment. Strategies and actions are urgently needed to improve parenting skills and mitigate the negative impact by strengthening the family environment.Funding Statement: National Natural Science Foundation of China, Shanghai Committee of Science and Technology, Shanghai Municipal Health Commission, Shanghai Education CommitteeDeclaration of Interests: We declare no competing interests.Ethics Approval Statement: The study was approved by the Institutional Review Board of the Shanghai Children’s Medical Center, the Shanghai Jiao Tong University School of Medicine (SCMCIRB-K2016022-01)