Recombinant Fc-fusion vaccine of RBD induced protection against SARS-CoV-2 in non-human primate and mice

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to infect people globally. The increased COVID-19 cases and no licensed vaccines highlight the need to develop safe and effective vaccines against SARS-CoV-2 infection. Multiple vaccines candidates are under pre-clinical or clinical trails with different strengths and weaknesses. Here we developed a pilot scale production of a recombinant subunit vaccine (RBD-Fc Vacc) with the Receptor Binding Domain of SARS-CoV-2 S protein fused with the Fc domain of human IgG1. RBD-Fc Vacc induced SARS-CoV-2 specific neutralizing antibodies in non-human primates and human ACE2 transgenic mice. The antibodies induced in macaca fascicularis neutralized three divergent SARS-CoV2 strains, suggesting a broader neutralizing ability. Three times immunizations protected Macaca fascicularis (20ug or 40ug per dose) and mice (10ug or 20ug per dose) from SARS-CoV-2 infection respectively. These data support clinical development of SARS-CoV-2 vaccines for humans. RBD-Fc Vacc is currently being assessed in randomized controlled phase 1/II human clinical trails. SummaryThis study confirms protective efficacy of a SARS-CoV-2 RBD-Fc subunit vaccine.

Rapid adaptation of SARS-CoV-2 in BALB/c mice: Novel mouse model for vaccine efficacy

Coronavirus disease 2019 (COVID-19) threatens global public health and economy. In order to develop safe and effective vaccines, suitable animal models must be established. Here we report the rapid adaption of SARS-CoV-2 in BALB/c mice, based on which a convenient, economical and effective animal model was developed. Specifically, we found that mouse-adapted SARS-CoV-2 at passage 6 (MACSp6) efficiently infected both aged and young wild-type BALB/c mice, resulting in moderate pneumonia as well as inflammatory responses. The elevated infectivity of MACSp6 in mice could be attributed to the substitution of a key residue (N501Y) in the receptorbinding domain (RBD). Using this novel animal model, we further evaluated the in vivo protective efficacy of an RBD-based SARS-CoV-2 subunit vaccine, which elicited highly potent neutralizing antibodies and conferred full protection against SARS-CoV-2 MACSp6 challenge. This novel mouse model is convenient and effective in evaluating the in vivo protective efficacy of SARS-CoV-2 vaccine. SummaryThis study describes a unique mouse model for SARS-CoV-2 infection and confirms protective efficacy of a SARS-CoV-2 RBD subunit vaccine.

Promotion of immune response by soluble Tim-3 in vitro and therapeutic potential / 军事医学

Objective To evaluate the role of recombinant human soluble Tim-3 (hTim-3-Fc) in regulating immune response.Methods Soluble hTim-3 was incubated with human macrophage cell line U 937, human T cell line Jurkat and normal human PBMC before cytokines secreted by or expressed in different immune cells were analyzed using ELISA , RT-PCR and Western-blotting, respectively.Results Soluble hTim-3 significantly promoted the activation of different immune cells.Our data showed that IL-8 secretion by U937 cells, IL-2 secretion by Jurkat cells , IL-2 and IFN-γsecretion by human PBMCs were all significantly increased .In addition , soluble hTim-3 significantly increased the IFN-α2 and IFN-β1 mRNA expression in U937, Jurkat and PBMCs and increased the phosphorylation of stat-1 in Jurkat and U937 cells.Conclusion Recombinant soluble hTim-3 can significantly promote the activation of immune cells in vitro, which shows its therapeutic potential .

Effect of estrogen on IL-10 production in mouse splenic B cells in vitro / 国际药学研究杂志

Objective To ascertain whether estrogen could induce B cell to produce interleukin(IL)-10. Methods C57BL/6 splenic B cells were purified by magnetic activated cell sorting(MACS)method,followed by estrogen treatment for 3 days. The secretion of IL-10 from cultured cell supernatant was tested by ELISA technique. The abundance of mRNA for IL-10、PD-L1 and RBM47 in B cells with estrogen treatment was tested by real-time PCR method. The intracellular IL-10 expression and the surface PD-L1 expression of treated B cells were tested by fluorescence activated cell sorting(FACS)method. And the expression of RBM47 in B cells by estrogen treatment was tested using Western blotting method. Results Estrogen could induce B cell to produce IL-10 in a dose-dependent manner. Estrogen treatment could increase the percentage of IL-10+B cells,the abundance of mRNA for IL-10,PD-L1 and RBM47 in B cells,as well as the expression of PD-L1 on B cell surface. Furthermore,our experimental result indicated the upreg?ulation of RBM47 expression in B cells by estrogen treatment. Conclusion Estrogen treatment in vitro can induce the upregulation of IL-10+regulatory B cells(Breg). Upregulation of RBM47 in the treated B cells might participate in this process by stabilizing IL-10 mRNA.

Roles of Tim-3 in paraquat poisoning induced acute inflammatory response / 军事医学

Objective To examine whether Tim-3 plays a protective role in paraquat poisoning induced excessive immune response and tissue damage based on the critical roles of Tim-3 controlling inflammatory response.Methods A paraquat poisoning model was established in wild type and in Tim-3 transgenic C57BL/6 mice by intraperitoneal injection of paraquat (40 mg/kg) .In addition, C57BL/6 mice with paraquat poisoning were injected with Tim-3 soluble protein( sTim-3) or control protein to see the effect of Tim-3 blocking on the progression of paraquat poisoning.Samples were collected at 6 and 24 h after paraquat injection respectively and were examined for tissue damage, cytokine expression and paraquat metabolism.Results After paraquat poisoning, there was significantly attenuated tissue damage in the lungs and kidneys and decreased TNF-α,IL-6 and IL-1 beta expression in the PBMCs or in the serum from Tim-3 transgenic mice compared to wild type mice.The serum concentration of paraquat in Tim-3 transgenic mice was also significantly decreased.However, in sTim-3 treated paraquat poisoning mice, there was significantly increased cytokine expression and tissue damage compared to control protein treated mice.The in vitro data showed that Tim-3 signaling negatively regulated macrophages mediated inflammatory response.Conclusion Tim-3 plays a critical role in maintaining the homeostasis after paraquat poisoning. Further investigation on the regulatory roles of Tim-3 in inflammation will shed new light on the pathogenesis of paraquat poisoning and provide new therapeutic strategies.

Death patterns of patients in Department of Respiratory Care Unit of General Hospi-tal of PLA / 军事医学

Objective To analyze retrospectively the death pattern, risk factors, and death time of 253 patients at the Respiratory Care Unit of General Hospital of PLA in order to improve care quality and reduce mortality.Methods The information of patients was extracted from the hospital information system ( HIS) , and then classified and calculated accord-ing to different time points.Results Between November and next March,the mortality rate was higher than in other months (P<0.05), accounting for 19.5%.Mortality of those admitted between 8∶01 and 9∶00 or between 23∶01 and 24∶00 was higher than at other times(P<0.05), accounting for 41.7%and 50.0%, respectively.There was statistically significant difference(P<0.01) in mortality between days of the week,with the highest on Saturday, accounting for 43.1%.Mortality on non-work days was higher than on workday(P<0.01), accounting for 38.3% and 13.2%, respectively.Mortality at off-hour was higher than at office time(8∶00-11∶30 and 14∶30-18∶00 on workday) (P<0.01), accounting for 31.3%and 5.2%, respectively.Logistic regression analysis showed that age, month of admission, and the hour of discharge were associated with the outcome.Conclusion The high mortality between November and next March may be related to the higher incidence of respiratory diseases in winter, air pollution and cold weather.High mortality is also significantly associ-ated with the care quality of the medical staff.

Antivirus mechanism of typeⅠinterferon:research progress / 军事医学

Interferon ( IFN) plays an essential role in antiviral infection.Interferons are divided into different categories according to their structure and function.People have attached increasing importance to TypeⅠinterferon( IFN-Ⅰ) in light of its unusual antiviral mechanism.This review is intended to shed light on IFN-Ⅰ,including its antiviral function,signal pathway and regulation.

Biological activity and application study of a monoclonal antibody against human Tim-3 / 军事医学

Objective To develop a human Tim-3 specific monoclonal antibody and evaluate its biological activity and possible use in clinical diseases associated with dysregulated Tim-3 expression .Methods The BALB/c mice were immu-nized by conventional method, and positive clones were used to develop anti-human Tim-3 antibody, the binding and neutralization activities of which in vitro and in vivo were investigated.Results ①A monoclonal antibody (clone L3D) which could specifically bind to human Tim-3 protein in ELISA assay was obtained and the subtype of the monoclonal antibody was IgG2a .②Flow cytometry indicated that the monoclonal antibody could bind to Tim-3 expressed in human U937 cells.This antibody also showed a cross activity to mice′Tim-3.③The monoclonal antibody inhibited the apoptosis of THP1 cells induced by Gal-9, the ligand of Tim-3.④Injection of Tim-3 antibody exacerbated sepsis in mice as marked by the decreased survival rate and increased expression of pro-inflammatory cytokines .Conclusion An anti-human Tim-3 monoclonal antibody is successfully obtained.The excellent binding and neutralization activities of this antibody enable it to be widely used in clinical diseases associated with deregulated Tim-3 expression .

A novel designed single domain antibody on 3-D structure of ricin A chain remarkably blocked ricin-induced cytotoxicity.

Ricin A chain (RA), an N-glycosidase, is able to fatally disrupt protein synthesis by attacking the Achilles heel of the ribosome RNA (rRNA). As specific immunotoxins, emergence of inhibitors for RA may obtain access to antagonistics against ricin intoxication and contribute to ameliorate the concomitant side effects. Many experimental results showed that the engineered VHs, which possessed solubility, stability, small size and consequently easier to express, purify and manipulate in vitro, were self and long-lived molecules compared to synthetic peptides. In this study, based on the crystal structure of RA, a novel recombinant human single-domain antibody expressing a polypeptide against RA in the CDR3 loop (named rVH(PT)) was obtained using computer-guided molecular design method. Theoretically, rVH(PT) could penetrate deeply into the active cleft of RA and act as a potent antagonist analogue to block the RA-rRNA interaction. Followed results showed that the recombinant VH(PT) was easily expressed of high-yield production and in a partially soluble fusion form in Escherichia coli. In vitro cytotoxicity experiments demonstrated that it possessed remarkable ability to block ricin-induced cytotoxicity. This study highlights the potential of human VH to display biostructure and biofunction of peptides designed on RA functional domain and could be useful in developing new antidotes with potential therapeutic uses to neutralize unintended exposure to ricin.

Progress in The Differentiation, Regulation and Function of Th17 Lineage / 生物化学与生物物理进展

As a new identified help T cell lineage different from Th1 and Th2 cells, Th17 cell has been found played important roles in the pathogenesis of autoimmunity and inflammatory disease. To further identify their roles, the differentiation and regulation of Th17 cells has been widely explored recently. Now it has been confirmed that TGF-beta, combined with IL-6 or IL-21, play critical roles in the differentiation of Th17 cells. While IL-23 mainly contribute in promoting the secretion of IL-17 and maintaining the function of Th17 cells. Corresponding with the Th1,Th2, and Treg cells, which has special transcription factors T-bet、GATA3、Foxp3 respectively, now it has been confirmed that ROR-?t(retinoid-related orphan receptors-?t) is the special transcription factor which specially regulate the differentiation of Th17 cells. Th17 cells function through their secreted pro-inflammatory cytokines, including IL-17A, IL-17F, IL-21, IL-22, IL-6, TNF-?. Among them IL-21,which act as a autocrine cytokine of Th17 cells, play critical roles in promoting the differentiation of Th17 cells while inhibiting the differentiation and function of Th1 and Treg cells. On the other hand, IL-2, which is obligatory for the growth of Th1,Th2,Treg and CD8+T cells, now has been found negatively regulate the differentiation of Th17 cells. In all, differentiation of Th17 and Treg,Th1 cells are exactly regulated in vivo, in which TGF-beta played critical roles. As both Th1 and Th17 cells participate in the pathogenesis of autoimmunity and inflammatory diseases, are they play synergistic roles or function at different time point or location? How TGF-? regulate Th17 and Treg cells?Can Th17 cells be used as a target for immune tolerance induction? All above questions will certainly be of continuing interest.

OBSERVATION OF THE DEVELOPMENT OF PLASMA MIP-1? AND MCP-1 IN RATS WITH PULMONARY FIBROSIS / 解放军医学杂志

To investigate the plasma MIP 1?and MCP 1 levels in rats with pulmonary fibrosis produced by bleomycin and preliminarily study the pathogenesis of the two chemokines in pulmonary fibrosis. The rats were randomly divided into 2 groups: normal control and pulmonary fibrotic group. Rats were killed on day 1, 3, 7, 14 and 28, and the plasma were collected .The solid phase sandwich enzyme linked immuno sorbent assay (ELISA) was used to assay the level of plasma MIP 1? and MCP 1. The results showed that the level of MIP 1? and MCP 1 in rats with pulmonary fibrosis were significantly higher than those in normal control ; plasma MCP 1 level was significantly correlated with plasma MIP 1?. It indicates that plasma MIP 1? and MCP 1 may be useful markers for monitoring the course of pulmonary fibrosis and they may reflect the clinical evidence of pulmonary fibrosis.

EXPRESSION OF HUMAN CD59 ANTIGEN ON MOUSE NIH3T3 AND EL-4 CELLS CONFERS PROTECTION AGAINST HUMAN COMPLEMENT ATTACK / 免疫学杂志

CD59 antigen is a widely expressed cell surface glycosylphosphatidyl-inositol (GPI) anchored glycoprotein.It acts as an inhibitor to the assembly of the membrane attack complex of homologous complement,binds to CD2,and also transduces activation signals with T cells.In this report,a 396bp DNA fragment was amplified by RT-PCR method from the total RNA of Jurkat cells.The fragment was cloned into pUC18 and pUC19 plas-mids,and further sequenced by Sanger′s-dideory-mediated chain termination.The results showed that this cDNA fragment included 384bp open reading fragment and its sequence was identical to the published sequence encoding human CD59 antigen.Furthermore,the cDNA of CD59 was subcloned into retroviral vector pLXSN and transfec-ted into packaging cell line PA317 to generate stable virus-producing cell lines.Then,mouse thymotase cell line EL-4 and fibroblasts cell line NIH3T3 were infected with the virus resulting in stable expression of CD59 on the cell surface.The transfected cells were tested for their susceptibility to human complement-mediated cytolysis.It was found that the transfected cells expressing CD59 antigen were far less susceptible than the controls,indicating that the gene for CD59 can be expressed in xenotypic cells stably to confer protection against human serum complement.