Comparative single-cell RNA sequencing analysis of immune response to inactivated vaccine and natural SARS-CoV-2 infection.

Uncovering the immune response to an inactivated SARS-CoV-2 vaccine (In-Vac) and natural infection is crucial for comprehending COVID-19 immunology. Here we conducted an integrated analysis of single-cell RNA sequencing (scRNA-seq) data from serial peripheral blood mononuclear cell (PBMC) samples derived from 12 individuals receiving In-Vac compared with those from COVID-19 patients. Our study reveals that In-Vac induces subtle immunological changes in PBMC, including cell proportions and transcriptomes, compared with profound changes for natural infection. In-Vac modestly upregulates IFN-α but downregulates NF-κB pathways, while natural infection triggers hyperactive IFN-α and NF-κB pathways. Both In-Vac and natural infection alter T/B cell receptor repertoires, but COVID-19 has more significant change in preferential VJ gene, indicating a vigorous immune response. Our study reveals distinct patterns of cellular communications, including a selective activation of IL-15RA/IL-15 receptor pathway after In-Vac boost, suggesting its potential role in enhancing In-Vac-induced immunity. Collectively, our study illuminates multifaceted immune responses to In-Vac and natural infection, providing insights for optimizing SARS-CoV-2 vaccine efficacy.

Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates.

Human respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract illness (LRTI), and no vaccine against LRTI has proven to be safe and effective in infants. Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice. The constructed recombinant plasmids harbored (5' to 3') a T7 promoter, hammerhead ribozyme, RSV Long strain antigenomic cDNA with cold-passaged (cp) mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain (A2cpts) or further combined with SH gene deletion (A2cptsΔSH), HDV ribozyme (δ), and a T7 terminator. These vectors were subsequently co-transfected with four helper plasmids encoding N, P, L, and M2-1 viral proteins into BHK/T7-9 cells, and the recovered viruses were then passaged in Vero cells. The rescued recombinant RSVs (rRSVs) were named rRSV-Long/A2cp, rRSV-Long/A2cpts, and rRSV-Long/A2cptsΔSH, respectively, and stably passaged in vitro, without reversion to wild type (wt) at sites containing introduced mutations or deletion. Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed  temperature-sensitive (ts) phenotype in vitro and in vivo, all rRSVs were significantly attenuated in vivo. Furthermore, BALB/c mice immunized with rRSVs produced Th1-biased immune response, resisted wtRSV infection, and were free from enhanced respiratory disease. We showed that the combination of ΔSH with attenuation (att) mutations of cpts contributed to improving att phenotype, efficacy, and gene stability of rRSV. By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains, we have laid an important foundation for the development of RSV live attenuated vaccines.

Inhibition of the B7-H3 immune checkpoint limits tumor growth by enhancing cytotoxic lymphocyte function.

The interaction between tumor and the immune system is still poorly understood. Significant clinical responses have been achieved in cancer patients treated with antibodies against the CTLA4 and PD-1/PD-L1 checkpoints; however, only a small portion of patients responded to the therapies, indicating a need to explore additional co-inhibitory molecules for cancer treatment. B7-H3, a member of the B7 superfamily, was previously shown by us to inhibit T-cell activation and autoimmunity. In this study, we have analyzed the function of B7-H3 in tumor immunity. Expression of B7-H3 was found in multiple tumor lines, tumor-infiltrating dendritic cells, and macrophages. B7-H3-deficient mice or mice treated with an antagonistic antibody to B7-H3 showed reduced growth of multiple tumors, which depended on NK and CD8+ T cells. With a putative receptor expressed by cytotoxic lymphocytes, B7-H3 inhibited their activation, and its deficiency resulted in increased cytotoxic lymphocyte function in tumor-bearing mice. Combining blockades of B7-H3 and PD-1 resulted in further enhanced therapeutic control of late-stage tumors. Taken together, our results indicate that the B7-H3 checkpoint may serve as a novel target for immunotherapy against cancer.

Isolation of murine hepatic lymphocytes using mechanical dissection for phenotypic and functional analysis of NK1.1+ cells.

AIM: To choose an appropriate methods for the isolation of hepatic lymphocytes between the mechanical dissection and the enzymatic digestion and investigate the effects of two methods on phenotype and function of hepatic lymphocytes. METHODS: Hepatic lymphocytes were isolated from untreated, poly (I:C)-stimulated or ConA-stimulated mice using the two methods, respectively. The cell yield per liver was evaluated by direct counting under microscope. Effects of digestive enzymes on the surface markers involved in hepatic lymphocytes were represented by relative change rate ((percentage of post-digestion -percentage of pre-digestion)/percentage of pre-digestion). Phenotypic analyses of the subpopulations of hepatic lymphocytes and intracellular cytokines were detected by flow cytometry. The cytotoxicity of NK cells from wild C57BL/6 or poly (I:C)-stimulated C57BL/6 mice was analyzed with a 4-h (51)Cr release assay. RESULTS: NK1.1(+) cell markers, NK1.1 and DX5, were significantly down-expressed after enzymatic digestion and their relative change rates were about 28% and 32%, respectively. Compared with the enzymatic digestion, the cell yield isolated from unstimulated, poly (I:C)-treated or ConA-treated mice by mechanical dissection was not significantly decreased. Hepatic lymphocytes isolated by the mechanical dissection comprised more innate immune cells like NK, NKT and gammadelta cells in normal C57BL/6 mice. After poly (I:C) stimulation, hepatic NK cells rose to about 35%, while NKT cells simultaneously decreased. Following ConA injection, the number of hepatic NKT cells was remarkably reduced to 3.67%. Higher ratio of intracellular IFN-gamma(+) (68%) or TNF-alpha(+) (15%) NK1.1(+) cells from poly (I:C)-treated mice was obtained using mechanical dissection method than control mice. There was no difference in viability between the mechanical dissection and the enzymatic digestion, and hepatic lymphocytes obtained with the two methods had similar cytotoxicity against YAC-1 cells. CONCLUSION: There is no difference in the cell yield and viability of the hepatic lymphocyte isolated with the two methods. The mechanical dissection, but not the enzymatic digestion, may be suitable for the phenotypic analysis of hepatic NK1.1(+) cell.