Respiratory immune status and microbiome in recovered COVID-19 patients revealed by metatranscriptomic analyses

Coronavirus disease 2019 (COVID-19) is currently a severe threat to global public health, and the immune response to COVID-19 infection has been widely investigated. However, the immune status and microecological changes in the respiratory systems of patients with COVID-19 after recovery have rarely been considered. We selected 72 patients with severe COVID-19 infection, 57 recovered from COVID-19 infection, and 65 with non-COVID-19 pneumonia, for metatranscriptomic sequencing and bioinformatics analysis. Accordingly, the differentially expressed genes between the infected and other groups were enriched in the chemokine signaling pathway, NOD-like receptor signaling pathway, phagosome, TNF signaling pathway, NF-kappa B signaling pathway, Toll-like receptor signaling pathway, and C-type lectin receptor signaling pathway. We speculate that IL17RD, CD74, and TNFSF15 may serve as disease biomarkers in COVID-19. Additionally, principal coordinate analysis revealed significant differences between groups. In particular, frequent co-infections with the genera Streptococcus, Veillonella, Gemella, and Neisseria, among others, were found in COVID-19 patients. Moreover, the random forest prediction model with differential genes showed a mean area under the curve (AUC) of 0.77, and KCNK12, IL17RD, LOC100507412, PTPRT, MYO15A, MPDZ, FLRT2, SPEG, SERPINB3, and KNDC1 were identified as the most important genes distinguishing the infected group from the recovered group. Agrobacterium tumefaciens, Klebsiella michiganensis, Acinetobacter pittii, Bacillus sp. FJAT.14266, Brevundimonas naejangsanensis, Pseudopropionibacterium propionicum, Priestia megaterium, Dialister pneumosintes, Veillonella rodentium, and Pseudomonas protegens were selected as candidate microbial markers for monitoring the recovery of COVID patients. These results will facilitate the diagnosis, treatment, and prognosis of COVID patients recovering from severe illness.

Determination of a reasonable threshold of total bilirubin for the diagnosis of hepatitis B virus- associated acute -on - chronic liver failure. (Theme Issue: New progress in the treatment of hepatobiliary tumors in the new era of immunotherapy.) [Chinese]

Objective: To investigate a reasonable threshold d total bilirubin for the diagnosis of hepatitis B virus - related acute - on -chronic liver failure (HBV - ACLF), and to realize accurate early diagnosis.

Lineage-mosaic and mutation-patched spike proteins for broad-spectrum COVID-19 vaccine (preprint)

The widespread SARS-CoV-2 in humans results in the continuous emergence of new variants. Recently emerged Omicron variant with multiple spike mutations sharply increases the risk of breakthrough infection or reinfection, highlighting the urgent need for new vaccines with broad-spectrum antigenic coverage. Using inter-lineage chimera and mutation patch strategies, we engineered a recombinant monomeric spike variant (STFK1628x), which showed high immunogenicity and mutually complementary antigenicity to its prototypic form (STFK). In hamsters, a bivalent vaccine comprised of STFK and STFK1628x elicited high titers of broad-spectrum antibodies to neutralize all 14 circulating SARS-CoV-2 variants, including Omicron; and fully protected vaccinees from intranasal SARS-CoV-2 challenges of either the ancestral strain or immune-evasive Beta variant. Strikingly, the vaccination of hamsters with the bivalent vaccine completely blocked the within-cage virus transmission to unvaccinated sentinels, for either the ancestral SARS-CoV-2 or Beta variant. Thus, our study provides new insights and antigen candidates for developing next-generation COVID-19 vaccines.

Sterilizing immunity against SARS-CoV-2 in hamsters conferred by a novel recombinant subunit vaccine (preprint)

A safe and effective SARS-CoV-2 vaccine is essential to avert the on-going COVID-19 pandemic. Here, we developed a subunit vaccine, which is comprised of CHO-expressed spike ectodomain protein (StriFK) and nitrogen bisphosphonates-modified zinc-aluminum hybrid adjuvant (FH002C). This vaccine candidate rapidly elicited the robust humoral response, Th1/Th2 balanced helper CD4 T cell and CD8 T cell immune response in animal models. In mice, hamsters, and non-human primates, 2-shot and 3-shot immunization of StriFK-FH002C generated 28- to 38-fold and 47- to 269-fold higher neutralizing antibody titers than the human COVID-19 convalescent plasmas, respectively. More importantly, the StriFK-FH002C immunization conferred sterilizing immunity to prevent SARS-CoV-2 infection and transmission, which also protected animals from virus-induced weight loss, COVID-19-like symptoms, and pneumonia in hamsters. Vaccine-induced neutralizing and cell-based receptor-blocking antibody titers correlated well with protective efficacy in hamsters, suggesting vaccine-elicited protection is immune-associated. The StriFK-FH002C provided a promising SARS-CoV-2 vaccine candidate for further clinical evaluation.

A comprehensive transcriptome analysis reveals broader but weaker host response of SARS-CoV-2 than SARS-CoV (preprint)

COVID-19, which has resulted a worldwide health crisis with more than 74.9 million confirmed cases worldwide by December 2020, is caused by a newly emerging coronavirus identified and named SARS-CoV-2 in February in Wuhan, China. Experiences in defeating SARS, which infested during 2002-2003, can be used in treating the new disease. However, comparative genomics and epidemiology studies have shown much difference between SARS-CoV and SARS-CoV-2, which underlies the different clinical features and therapies in between those two diseases. Further studies comparing transcriptomes infected by these two viruses to uncover the differences in host responses would be necessary. Here we conducted a comprehensive transcriptome analysis of SARS-CoV and SARS-CoV-2-infected human cell lines, including Caco-2, Calu-3, H1299. Clustering analysis and expression of ACE2 show that SARS-CoV-2 has broader but weaker infection, where the largest discrepancy occurs in the epithelial lung cancer cell, Calu-3. SARS-CoV-2 genes also show less tissue specificity than SARS-CoV genes. Furthermore, we detected more general but moderate immune responses in SARS-CoV-2 infected transcriptomes by comparing weighted gene co-expression networks and modules. Our results suggest a different immune therapy and treatment scheme for COVID-19 patients than the ones used on SARS patients. The wider but weaker permissiveness and host responses of virus infection may also imply a long-term existence of SARS-CoV-2 among human populations.

Decreased T Cell Levels in Critically Ill Coronavirus Patients: single-center, prospective and observational study (preprint)

Background: Since Dec. 2019, COVID-19 pandemic has been outbreak. T cells play an important role in dealing with various disease-causing pathogens. However, the role of T cells played in COVID-19 patients is still unknown. Our study aimed to describe immunologic state of the critical ill COVID-19 patients. Methods: 63 patients with confirmed COVID-19 pneumonia admitted Department of Intensive Care Unit of the First Affiliated Hospital of Harbin Medical University. The immunologic characteristics（lymphocyte apoptosis, the expression of PD-1 and HLA-DR in T cells, T cell subset levels, redistribution and the production of inflammatory factors）as well as their laboratory parameters were compared between severe group and critical group.Results: The level of T cells in peripheral blood was decreased in critical patients compared with that in severe patients, but the expression levels of PD-1 (CD4+: 24.71% VS 30.56%; CD8+: 33.05% VS 32.38%) and HLA-DR (T cells: 36.28% VS 27.44%; monocytes: 20.58% VS 23.83%) in T cells were not significantly changed, and apoptosis and necrosis were not different in lymphocytes (apoptosis: 1.04% VS 1.27%; necrosis: 0.67% VS 1.11%), granulocytes, or monocytes between those two groups.Conclusions: There is severe immunosuppression in critical ill COVID-19 patients. Redistribution of T cells might be the main reason for lymphocytic decline. Decreasing the infiltration of T lymphocytes in the lung may be beneficial for the treatment of COVID-19. Trial registration: The study was approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University. Code number: kyk2020003. 

Virus-free and live-cell visualizing SARS-CoV-2 cell entry for studies of neutralizing antibodies and compound inhibitors (preprint)

The ongoing COVID-19 pandemic, caused by SARS-CoV-2 infection, has resulted in hundreds of thousands of deaths. Cellular entry of SARS-CoV-2, which is mediated by the viral spike protein and host ACE2 receptor, is an essential target for the development of vaccines, therapeutic antibodies, and drugs. Using a mammalian cell expression system, we generated a recombinant fluorescent protein (Gamillus)-fused SARS-CoV-2 spike trimer (STG) to probe the viral entry process. In ACE2-expressing cells, we found that the STG probe has excellent performance in the live-cell visualization of receptor binding, cellular uptake, and intracellular trafficking of SARS-CoV-2 under virus-free conditions. The new system allows quantitative analyses of the inhibition potentials and detailed influence of COVID-19-convalescent human plasmas, neutralizing antibodies and compounds, providing a versatile tool for high-throughput screening and phenotypic characterization of SARS-CoV-2 entry inhibitors. This approach may also be adapted to develop a viral entry visualization system for other viruses.

Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells (preprint)

The global pandemic of Coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable in vitro neutralization assay is very important for the development of neutralizing antibodies, vaccines and other inhibitors. In this study, G protein-deficient vesicular stomatitis virus (VSVdG) bearing full-length and truncated spike (S) protein of SARS-CoV-2 were evaluated. The virus packaging efficiency of VSV-SARS-CoV-2-Sdel18 (S with C-terminal 18 amino acid truncation) is much higher than VSV-SARS-CoV-2-S. A neutralization assay for antibody screening and serum neutralizing titer quantification was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and human angiotensin-converting enzyme 2 (ACE2) overexpressed BHK21 cell (BHK21-hACE2). The experimental results can be obtained by automatically counting EGFP positive cell number at 12 hours after infection, making the assay convenient and high-throughput. The serum neutralizing titer of COVID-19 convalescent patients measured by VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with live SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting receptor binding domain (RBD) of SARS-CoV-2-S were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.