A triple-RBD-based mucosal vaccine provides broad protection against SARS-CoV-2 variants of concern.

The rapid mutation and spread of SARS-CoV-2 variants urge the development of effective mucosal vaccines to provide broad-spectrum protection against the initial infection and thereby curb the transmission potential. Here, we designed a chimeric triple-RBD immunogen, 3Ro-NC, harboring one Delta RBD and two Omicron RBDs within a novel protein scaffold. 3Ro-NC elicits potent and broad RBD-specific neutralizing immunity against SARS-CoV-2 variants of concern. Notably, intranasal immunization with 3Ro-NC plus the mucosal adjuvant KFD (3Ro-NC + KFDi.n) elicits coordinated mucosal IgA and higher neutralizing antibody specificity (closer antigenic distance) against the Omicron variant. In Omicron-challenged human ACE2 transgenic mice, 3Ro-NC + KFDi.n immunization significantly reduces the tissue pathology in the lung and lowers the viral RNA copy numbers in both the lung (85.7-fold) and the nasal turbinate (13.6-fold). Nasal virologic control is highly correlated with RBD-specific secretory IgA antibodies. Our data show that 3Ro-NC plus KFD is a promising mucosal vaccine candidate for protection against SARS-CoV-2 Omicron infection, pathology and transmission potential.

Bruton tyrosine kinase inhibitors in B-cell lymphoma: beyond the antitumour effect.

Targeting B-cell receptor signalling using Bruton tyrosine kinase (BTK) inhibitors (BTKis) has become a highly successful treatment modality for B-cell malignancies, especially for chronic lymphocytic leukaemia. However, long-term administration of BTKis can be complicated by adverse on- and/or off-target effects in particular cell types. BTK is widely expressed in cells of haematopoietic origin, which are pivotal components of the tumour microenvironment. BTKis, thus, show broad immunomodulatory effects on various non-B immune cell subsets by inhibiting specific immune receptors, including T-cell receptor and Toll-like receptors. Furthermore, due to the off-target inhibition of other kinases, such as IL-2-inducible T-cell kinase, epidermal growth factor receptor, and the TEC and SRC family kinases, BTKis have additional distinct effects on T cells, natural killer cells, platelets, cardiomyocytes, and other cell types. Such mechanisms of action might contribute to the exceptionally high clinical efficacy as well as the unique profiles of adverse effects, including infections, bleeding, and atrial fibrillation, observed during BTKi administration. However, the immune defects and related infections caused by BTKis have not received sufficient attention in clinical studies till date. The broad involvement of BTK in immunological pathways provides a rationale to combine BTKis with specific immunotherapies, such as immune checkpoint inhibitor or chimeric antigen receptor-T-cell therapy, for the treatment of relapsed or refractory diseases. This review discusses and summarises the above-mentioned issues as a reference for clinicians and researchers.

Comparative global B cell receptor repertoire difference induced by SARS-CoV-2 infection or vaccination via single-cell V(D)J sequencing.

Dynamic changes of the paired heavy and light chain B cell receptor (BCR) repertoire provide an essential insight into understanding the humoral immune response post-SARS-CoV-2 infection and vaccination. However, differences between the endogenous paired BCR repertoire kinetics in SARS-CoV-2 infection and previously recovered/naïve subjects treated with the inactivated vaccine remain largely unknown. We performed single-cell V(D)J sequencing of B cells from six healthy donors with three shots of inactivated SARS-CoV-2 vaccine (BBIBP-CorV), five people who received the BBIBP-CorV vaccine after having recovered from COVID-19, five unvaccinated COVID-19 recovered patients and then integrated with public data of B cells from four SARS-CoV-2-infected subjects. We discovered that BCR variable (V) genes were more prominently used in the SARS-CoV-2 exposed groups (both in the group with active infection and in the group that had recovered) than in the vaccinated groups. The VH gene that expanded the most after SARS-CoV-2 infection was IGHV3-33, while IGHV3-23 in the vaccinated groups. SARS-CoV-2-infected group enhanced more BCR clonal expansion and somatic hypermutation than the vaccinated healthy group. A small proportion of public clonotypes were shared between the SARS-CoV-2 infected, vaccinated healthy, and recovered groups. Moreover, several public antibodies had been identified against SARS-CoV-2 spike protein. We comprehensively characterize the paired heavy and light chain BCR repertoire from SARS-CoV-2 infection to vaccination, providing further guidance for the development of the next-generation precision vaccine.

Codelivery of SARS-CoV-2 Prefusion-Spike Protein with CBLB502 by a Dual-Chambered Ferritin Nanocarrier Potentiates Systemic and Mucosal Immunity.

Thousands of breakthrough infections are confirmed after intramuscular (i.m.) injection of the approved vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Two major factors might contribute to breakthrough infections. One is the emergence of mutant variants of SARS-CoV-2, and the other is that i.m. injection has an inefficient ability to activate mucosal immunity in the upper respiratory tract. Here, we devised a dual-chambered nanocarrier that can codeliver the adjuvant CBLB502 with prefusion-spike (pre-S) onto a ferritin nanoparticle. This vaccine enabled enhanced systemic and local mucosal immunity in the upper and lower respiratory tract. Further, codelivery of CBLB502 with pre-S induced a Th1/Th2-balanced immunoglobulin G response. Moreover, the codelivery nanoparticle showed a Th1-biased cellular immune response as the release of splenic INF-γ was significantly heightened while the level of IL-4 was elevated to a moderate extent. In general, the developed dual-chambered nanoparticle can trigger multifaceted immune responses and shows great potential for mucosal vaccine development.

Structure-Based Discovery of Novel Nonpeptide Inhibitors Targeting SARS-CoV-2 Mpro.

The continual spread of novel coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), posing a severe threat to the health worldwide. The main protease (Mpro, alias 3CLpro) of SARS-CoV-2 is a crucial enzyme for the maturation of viral particles and is a very attractive target for designing drugs to treat COVID-19. Here, we propose a multiple conformation-based virtual screening strategy to discover inhibitors that can target SARS-CoV-2 Mpro. Based on this strategy, nine Mpro structures and a protein mimetics library with 8960 commercially available compounds were prepared to carry out ensemble docking for the first time. Five of the nine structures are apo forms presented in different conformations, whereas the other four structures are holo forms complexed with different ligands. The surface plasmon resonance assay revealed that 6 out of 49 compounds had the ability to bind to SARS-CoV-2 Mpro. The fluorescence resonance energy transfer experiment showed that the biochemical half-maximal inhibitory concentration (IC50) values of the six compounds could hamper Mpro activities ranged from 0.69 ± 0.05 to 2.05 ± 0.92 µM. Evaluation of antiviral activity using the cell-based assay indicated that two compounds (Z1244904919 and Z1759961356) could strongly inhibit the cytopathic effect and reduce replication of the living virus in Vero E6 cells with the half-maximal effective concentrations (EC50) of 4.98 ± 1.83 and 8.52 ± 0.92 µM, respectively. The mechanism of the action for the two inhibitors were further elucidated at the molecular level by molecular dynamics simulation and subsequent binding free energy analysis. As a result, the discovered noncovalent reversible inhibitors with novel scaffolds are promising antiviral drug candidates, which may be used to develop the treatment of COVID-19.

Rapid isolation and immune profiling of SARS-CoV-2 specific memory B cell in convalescent COVID-19 patients via LIBRA-seq.

B cell response plays a critical role against SARS-CoV-2 infection. However, little is known about the diversity and frequency of the paired SARS-CoV-2 antigen-specific BCR repertoire after SARS-CoV-2 infection. Here, we performed single-cell RNA sequencing and VDJ sequencing using the memory and plasma B cells isolated from five convalescent COVID-19 patients, and analyzed the spectrum and transcriptional heterogeneity of antibody immune responses. Via linking BCR to antigen specificity through sequencing (LIBRA-seq), we identified a distinct activated memory B cell subgroup (CD11chigh CD95high) had a higher proportion of SARS-CoV-2 antigen-labeled cells compared with memory B cells. Our results revealed the diversity of paired BCR repertoire and the non-stochastic pairing of SARS-CoV-2 antigen-specific immunoglobulin heavy and light chains after SARS-CoV-2 infection. The public antibody clonotypes were shared by distinct convalescent individuals. Moreover, several antibodies isolated by LIBRA-seq showed high binding affinity against SARS-CoV-2 receptor-binding domain (RBD) or nucleoprotein (NP) via ELISA assay. Two RBD-reactive antibodies C14646P3S and C2767P3S isolated by LIBRA-seq exhibited high neutralizing activities against both pseudotyped and authentic SARS-CoV-2 viruses in vitro. Our study provides fundamental insights into B cell response following SARS-CoV-2 infection at the single-cell level.

Computational design and modeling of nanobodies toward SARS-CoV-2 receptor binding domain.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global health concern and pose a serious threat to humanity. There is an urgent need for developing therapeutic drugs and (or) biologics to prevent the spread of the virus. The life cycle of SARS-CoV-2 shows that the virus enters host cells by first binding to angiotensin-converting enzyme 2 (ACE2) through its spike protein receptor-binding domain (RBD). Therefore, blocking the binding between of ACE2 and SARS-CoV-2 RBD can inhibit the virus infection in the host cells. In this study, by grafting the complementarity-determining regions (CDRs) of developed SARS-CoV, MERS-CoVs specific neutralizing antibodies (nAbs) include monoclonal antibodies (mAbs) as well as SARS-CoV-2 mAbs onto a known stable nanobody (Nb) scaffold, and a total of 16 Nbs sequences were designed. Five Nbs, namely CS01, CS02, CS03, CS10, and CS16, were selected based on the free energy landscape of protein docking verified by the recently reported Nb-RBD cocrystal structures. CS01, CS02, and CS03 occupied the ACE2 binding site of RBD, while CS10 and CS16 were proposed to inhibit the interaction between RBD and ACE2 through an allosteric mechanism. Based on the structures of the five Nbs in complex with RBD, seven brand-new Nbs with enhanced binding affinities (CS02_RD01, CS03_RD01, CS03_RD02, CS03_RD03, CS03_RD04, CS16_RD01, and CS16_RD02) were generated by redesign of residues on the interface of the five Nbs contact with SARS-CoV-2 RBD. In addition, the identified "hot spots" on the interface of each complex provide useful information to understand the binding mechanism of designed Nbs to SARS-CoV-2 RBD. In sum, the predicted stabilities and high binding affinities of the 11 (re)designed Nbs indicating the potential of the developed computational framework in this work to design effective agents to block the infection of SARS-CoV-2.

Broad phenotypic alterations and potential dysfunction of lymphocytes in individuals clinically recovered from COVID-19.

Although millions of patients have clinically recovered from COVID-19, little is known about the immune status of lymphocytes in these individuals. In this study, the peripheral blood mononuclear cells of a clinically recovered (CR) cohort were comparatively analyzed with those of an age- and sex-matched healthy donor cohort. We found that CD8+ T cells in the CR cohort had higher numbers of effector T cells and effector memory T cells but lower Tc1 (IFN-γ+), Tc2 (IL-4+), and Tc17 (IL-17A+) cell frequencies. The CD4+ T cells of the CR cohort were decreased in frequency, especially the central memory T cell subset. Moreover, CD4+ T cells in the CR cohort showed lower programmed cell death protein 1 (PD-1) expression and had lower frequencies of Th1 (IFN-γ+), Th2 (IL-4+), Th17 (IL-17A+), and circulating follicular helper T (CXCR5+PD-1+) cells. Accordingly, the proportion of isotype-switched memory B cells (IgM-CD20hi) among B cells in the CR cohort showed a significantly lower proportion, although the level of the activation marker CD71 was elevated. For CD3-HLA-DR- lymphocytes in the CR cohort, in addition to lower levels of IFN-γ, granzyme B and T-bet, the correlation between T-bet and IFN-γ was not observed. Additionally, by taking into account the number of days after discharge, all the phenotypes associated with reduced function did not show a tendency toward recovery within 4‒11 weeks. The remarkable phenotypic alterations in lymphocytes in the CR cohort suggest that  severe acute respiratory syndrome coronavirus 2 infection profoundly affects lymphocytes and potentially results in dysfunction even after clinical recovery.

Characteristics of T-cell responses in COVID-19 patients with prolonged SARS-CoV-2 positivity - a cohort study.

OBJECTIVE: SARS-CoV-2 has caused a worldwide pandemic of COVID-19. The existence of prolonged SARS-CoV-2 positivity (PP) has further increased the burden on the health system. Since T cells are vital for viral control, we aimed to evaluate the characteristics of T-cell responses associated with PP. METHODS: We established a PP cohort and two age- and sex-matched control cohorts: a regular clinical recovery (CR) cohort and a healthy donor (HD) cohort. The mean time for RNA negativity conversion in the PP cohort was markedly longer than that in the CR cohort (66.2 vs 25.3 days), while the time from illness onset to sampling was not significantly different. T-cell responses in the PP cohort were assayed, analysed and compared with those in the CR and HD cohorts by flow cytometry and ELISpot analysis of peripheral blood mononuclear cells. RESULTS: Compared with the CR cohort, the proliferation, activation and functional potential of CD8+ and CD4+ T cells in the PP cohort were not significantly different. However, the frequencies and counts of Teff and Tem in CD8+ but not in CD4+ T cells of the PP cohort were prominently lower. Moreover, a weaker SARS-CoV-2 N protein-specific IFN-γ+ T-cell response and a higher frequency of Tregs were detected in the PP cohort. CONCLUSION: Suppressed CD8+ T-cell differentiation is associated with PP and may be an indicator for the prediction of prolonged SARS-CoV-2 positivity in COVID-19 patients. The association between suppressed CD8+ T-cell differentiation and elevated Tregs warrants studies in the future.

A deep learning algorithm using CT images to screen for Corona virus disease (COVID-19).

OBJECTIVE: The outbreak of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-COV-2) has caused more than 26 million cases of Corona virus disease (COVID-19) in the world so far. To control the spread of the disease, screening large numbers of suspected cases for appropriate quarantine and treatment are a priority. Pathogenic laboratory testing is typically the gold standard, but it bears the burden of significant false negativity, adding to the urgent need of alternative diagnostic methods to combat the disease. Based on COVID-19 radiographic changes in CT images, this study hypothesized that artificial intelligence methods might be able to extract specific graphical features of COVID-19 and provide a clinical diagnosis ahead of the pathogenic test, thus saving critical time for disease control. METHODS: We collected 1065 CT images of pathogen-confirmed COVID-19 cases along with those previously diagnosed with typical viral pneumonia. We modified the inception transfer-learning model to establish the algorithm, followed by internal and external validation. RESULTS: The internal validation achieved a total accuracy of 89.5% with a specificity of 0.88 and sensitivity of 0.87. The external testing dataset showed a total accuracy of 79.3% with a specificity of 0.83 and sensitivity of 0.67. In addition, in 54 COVID-19 images, the first two nucleic acid test results were negative, and 46 were predicted as COVID-19 positive by the algorithm, with an accuracy of 85.2%. CONCLUSION: These results demonstrate the proof-of-principle for using artificial intelligence to extract radiological features for timely and accurate COVID-19 diagnosis. KEY POINTS: • The study evaluated the diagnostic performance of a deep learning algorithm using CT images to screen for COVID-19 during the influenza season. • As a screening method, our model achieved a relatively high sensitivity on internal and external CT image datasets. • The model was used to distinguish between COVID-19 and other typical viral pneumonia, both of which have quite similar radiologic characteristics.

Alterations in Phenotypes and Responses of T Cells Within 6 Months of Recovery from COVID-19: A Cohort Study.

The COVID-19 pandemic, caused by the SARS-CoV-2 infection, is a global health crisis. While many patients have clinically recovered, little is known about long-term alterations in T cell responses of COVID-19 convalescents. In this study, T cell responses in peripheral blood mononuclear cells of a long-time COVID-19 clinically recovered (20-26 weeks) cohort (LCR) were measured via flow cytometry and ELISpot. The T cell responses of LCR were comparatively analyzed against an age and sex matched short-time clinically recovered (4-9 weeks) cohort (SCR) and a healthy donor cohort (HD). All volunteers were recruited from Wuhan Jinyintan Hospital, China. Phenotypic analysis showed that activation marker PD-1 expressing on CD4+ T cells of LCR was still significantly lower than that of HD. Functional analysis indicated that frequencies of Tc2, Th2 and Th17 in LCR were comparable to those of HD, but Tc17 was higher than that of HD. In LCR, compared to the HD, there were fewer IFN-γ producing T cells but more IL-2 secreting T cells. In addition, the circulating Tfh cells in LCR were still slightly lower compared to HD, though the subsets composition had recovered. Remarkably, SARS-CoV-2 specific T cell responses in LCR were comparable to that of SCR. Collectively, T cell responses experienced long-term alterations in phenotype and functional potential of LCR cohort. However, after clinical recovery, SARS-CoV-2 specific T cell responses could be sustained at least for six months, which may be helpful in resisting re-infection.

A Lightweight Internet Sharing Scheme for Sectional Medical Images according to Existing Hospital Network Facilities and Basic Information Security Rules.

Background: With the outbreak of COVID-19, large-scale telemedicine applications can play an important role in the epidemic areas or less developed areas. However, the transmission of hundreds of megabytes of Sectional Medical Images (SMIs) from hospital's Intranet to the Internet has the problems of efficiency, cost, and security. This article proposes a novel lightweight sharing scheme for permitting Internet users to quickly and safely access the SMIs from a hospital using an Internet computer anywhere but without relying on a virtual private network or another complex deployment. Methods: A four-level endpoint network penetration scheme based on the existing hospital network facilities and information security rules was proposed to realize the secure and lightweight sharing of SMIs over the Internet. A "Master-Slave" interaction to the interactive characteristics of multiplanar reconstruction and maximum/minimum/average intensity projection was designed to enhance the user experience. Finally, a prototype system was established. Results: When accessing SMIs with a data size ranging from 251.6 to 307.04 MB with 200 kBps client bandwidth (extreme test), the network response time to each interactive request remained at approximately 1 s, the original SMIs were kept in the hospital, and the deployment did not require a complex process; the imaging quality and interactive experience were recognized by radiologists. Conclusions: This solution could serve Internet medicine at a low cost and may promote the diversified development of mobile medical technology. Under the current COVID-19 epidemic situation, we expect that it could play a low-cost and high-efficiency role in remote emergency support.