A major challenge to end the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is to develop a broadly protective vaccine that elicits long-term immunity. As the key immunogen, the viral surface spike (S) protein is frequently mutated, and conserved epitopes are shielded by glycans. Here, we revealed that S protein glycosylation has site-differential effects on viral infectivity. We found that S protein generated by lung epithelial cells has glycoforms associated with increased infectivity. Compared to the fully glycosylated S protein, immunization of S protein with N-glycans trimmed to the mono-GlcNAc–decorated state (S _MG ) elicited stronger immune responses and better protection for human angiotensin-converting enzyme 2 (hACE2) transgenic mice against variants of concern (VOCs). In addition, a broadly neutralizing monoclonal antibody was identified from S _MG -immunized mice that could neutralize wild-type SARS-CoV-2 and VOCs with subpicomolar potency. Together, these results demonstrate that removal of glycan shields to better expose the conserved sequences has the potential to be an effective and simple approach for developing a broadly protective SARS-CoV-2 vaccine.

Background. The Delta and Omicron variants of SARS-CoV-2 are currently responsible for breakthrough infections due to waning immunity. We report phase I/II trial results of UB-612, a multitope subunit vaccine containing S1-RBD-sFc protein and rationally designed promiscuous peptides representing sarbecovirus conserved helper T cell and cytotoxic T lymphocyte epitopes on the nucleocapsid (N), membrane (M), and spike (S2) proteins. Method. We conducted a phase I primary 2-dose (28 days apart) trial of 10, 30, or 100 μg UB-612 in 60 healthy young adults 20 to 55 years old, and 50 of them were boosted with 100 μg of UB-612 approximately 7 to 9 months after the second dose. A separate placebo-controlled and randomized phase II study was conducted with 2 doses of 100 μg of UB-612 (n = 3,875, 18–85 years old). We evaluated interim safety and immunogenicity of phase I until 14 days after the third (booster) dose and of phase II until 28 days after the second dose. Results. No vaccine-related serious adverse events were recorded. The most common solicited adverse events were injection site pain and fatigue, mostly mild and transient. In both trials, UB-612 elicited respective neutralizing antibody titers similar to a panel of human convalescent sera. The most striking findings were long-lasting virus-neutralizing antibodies and broad T cell immunity against SARS-CoV-2 variants of concern (VoCs), including Delta and Omicron, and a strong booster-recalled memory immunity with high cross-reactive neutralizing titers against the Delta and Omicron VoCs. Conclusion. UB-612 has presented a favorable safety profile, potent booster effect against VoCs, and long-lasting B and broad T cell immunity that warrants further development for both primary immunization and heterologous boosting of other COVID-19 vaccines. Trial Registration. ClinicalTrials.gov: NCT04545749, NCT04773067, and NCT04967742. Funding. UBI Asia, Vaxxinity Inc., and Taiwan Centers for Disease Control, Ministry of Health and Welfare.

Accelerated cell apoptosis with dysregulated long noncoding RNAs is the crucial pathogenesis in lupus nephritis (LN). Pro-apoptotic lincRNA-p21 was studied in LN patients, cell lines with lentivirus-mediated overexpression and CRISPR interference (CRISPRi)-conducted repression, and a mouse model. Clinical samples were from patients and age/sex-matched controls. Expression of lincRNA-p21 and endogenous RNA target miR-181a, were examined in mononuclear and urine cells. Guide RNA sequences targeting lincRNA-p21 were cloned into CRISPRi with dCas9/ Krüppel-associated box (KRAB) domain. LincRNA-p21-silened transfectants were investigated for apoptosis and miR-181a expression. LincRNA-p21-overexpressed cells were evaluated for apoptosis and p53-related down-stream molecules. Balb/C mice were injected with pristane to induce LN and examined for apoptosis and lincRNA-p21. Higher lincRNA-p21 levels were found in LN mononuclear and urine cells, positively correlated with activity. There were lower miR-181a levels in LN mononuclear cells, negatively correlated with activity. Doxorubicin-induced apoptotic cells had up-regulated lincRNA-p21 levels. CRISPRi with dCas9/KARA domain showed efficient repression ability on transcription initiation/elongation. CRISPRi-conducted lincRNA-p21-silenced transfectants displayed reduced apoptosis with up-regulated miR-181a levels, whereas lentivirus-mediated lincRNA-p21-overexpressed cells revealed enhanced apoptosis with up-regulated downstream PUMA/Bax expression. LN mice had glomerular apoptosis with progressive increased lincRNA-p21 levels. Our results demonstrate up-regulated lincRNA-p21 expression in LN, implicating a potential diagnostic marker and therapeutic target.

Genome-wide RNA interference (RNAi) screening is an emerging and powerful technique for genetic screens, which can be divided into arrayed RNAi screen and pooled RNAi screen/selection based on different screening strategies. To date, several genome-wide RNAi screens have been successfully performed to identify host factors essential for influenza virus replication. However, the host factors identified by different research groups are not always consistent. Taking influenza virus screens as an example, we found that a number of screening parameters may directly or indirectly influence the primary hits identified by the screens. This review highlights the differences among the published genome-wide screening approaches and offers recommendations for performing a good pooled shRNA screen/selection.

COVID-19 in humans is caused by Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that belongs to the beta family of coronaviruses. SARS-CoV-2 causes severe respiratory illness in 10–15% of infected individuals and mortality in 2–3%. Vaccines are urgently needed to prevent infection and to contain viral spread. Although several mRNA- and adenovirus-based vaccines are highly effective, their dependence on the “cold chain” transportation makes global vaccination a difficult task. In this context, a stable lyophilized vaccine may present certain advantages. Accordingly, establishing additional vaccine platforms remains vital to tackle SARS-CoV-2 and any future variants that may arise. Vaccinia virus (VACV) has been used to eradicate smallpox disease, and several attenuated viral strains with enhanced safety for human applications have been developed. We have generated two candidate SARS-CoV-2 vaccines based on two vaccinia viral strains, MVA and v-NY, that express full-length SARS-CoV-2 spike protein. Whereas MVA is growth-restricted in mammalian cells, the v-NY strain is replication-competent. We demonstrate that both candidate recombinant vaccines induce high titers of neutralizing antibodies in C57BL/6 mice vaccinated according to prime-boost regimens. Furthermore, our vaccination regimens generated T_H1-biased immune responses in mice. Most importantly, prime-boost vaccination of a Syrian hamster infection model with MVA-S and v-NY-S protected the hamsters against SARS-CoV-2 infection, supporting that these two vaccines are promising candidates for future development. Finally, our vaccination regimens generated neutralizing antibodies that partially cross-neutralized SARS-CoV-2 variants of concern.

Diffuse alveolar hemorrhage (DAH) in systemic lupus erythematosus (SLE) is associated with significant mortality, requiring a thorough understanding of its complex mechanisms to develop novel therapeutics for disease control. Activated p53-dependent apoptosis with dysregulated long non-coding RNA (lncRNA) expression is involved in the SLE pathogenesis and correlated with clinical activity. We examined the expression of apoptosis-related p53-dependent lncRNA, including H19, HOTAIR and lincRNA-p21 in SLE-associated DAH patients. Increased lincRNA-p21 levels were detected in circulating mononuclear cells, mainly in CD4+ and CD14+ cells. Higher expression of p53, lincRNA-p21 and cell apoptosis was identified in lung tissues. Lentivirus-based short hairpin RNA (shRNA)-transduced stable transfectants were created for examining the targeting efficacy in lncRNA. Under pristane stimulation, alveolar epithelial cells had increased p53, lincRNA-p21 and downstream Bax levels with elevated apoptotic ratios. After pristane injection, C57/BL6 mice developed DAH with increased pulmonary expression of p53, lincRNA-p21 and cell apoptosis. Intra-pulmonary delivery of shRNA targeting lincRNA-p21 reduced hemorrhage frequencies and improved anemia status through decreasing Bax expression and cell apoptosis. Our findings demonstrate increased p53-dependent lncRNA expression with accelerated cell apoptosis in the lungs of SLE-associated DAH patients, and show the therapeutic potential of targeting intra-pulmonary lncRNA expression in a pristane-induced model of DAH.