ORC1 enhances repressive epigenetic modifications on HIV-1 LTR to promote HIV-1 latency.

The human immunodeficiency virus type 1 (HIV-1) reservoir consists of latently infected cells which present a major obstacle to achieving a functional cure for HIV-1. The formation and maintenance of HIV-1 latency have been extensively studied, and latency-reversing agents (LRAs) that can reactivate latent HIV-1 by targeting the involved host factors are developed; however, their clinical efficacies remain unsatisfactory. Therefore, it is imperative to identify novel targets for more potential candidates or better combinations for LRAs. In this study, we utilized CRISPR affinity purification in situ of regulatory elements system to screen for host factors associated with the HIV-1 long terminal repeat region that could potentially be involved in HIV-1 latency. We successfully identified that origin recognition complex 1 (ORC1), the largest subunit of the origin recognition complex, contributes to HIV-1 latency in addition to its function in DNA replication initiation. Notably, ORC1 is enriched on the HIV-1 promoter and recruits a series of repressive epigenetic elements, including DNMT1 and HDAC1/2, and histone modifiers, such as H3K9me3 and H3K27me3, thereby facilitating the establishment and maintenance of HIV-1 latency. Moreover, the reactivation of latent HIV-1 through ORC1 depletion has been confirmed across various latency cell models and primary CD4+ T cells from people living with HIV-1. Additionally, we comprehensively validated the properties of liquid-liquid phase separation (LLPS) of ORC1 from multiple perspectives and identified the key regions that promote the formation of LLPS. This property is important for the recruitment of ORC1 to the HIV-1 promoter. Collectively, these findings highlight ORC1 as a potential novel target implicated in HIV-1 latency and position it as a promising candidate for the development of novel LRAs. IMPORTANCE: Identifying host factors involved in maintaining human immunodeficiency virus type 1 (HIV-1) latency and understanding their mechanisms prepares the groundwork to discover novel targets for HIV-1 latent infection and provides further options for the selection of latency-reversing agents in the "shock" strategy. In this study, we identified a novel role of the DNA replication factor origin recognition complex 1 (ORC1) in maintaining repressive chromatin structures surrounding the HIV-1 promoter region, thereby contributing to HIV-1 latency. This discovery expands our understanding of the non-replicative functions of the ORC complex and provides a potential therapeutic strategy for HIV-1 cure.

The construction of modular universal chimeric antigen receptor T (MU-CAR-T) cells by covalent linkage of allogeneic T cells and various antibody fragments.

BACKGROUND: Chimeric antigen receptor-T (CAR-T) cells therapy is one of the novel immunotherapeutic approaches with significant clinical success. However, their applications are limited because of long preparation time, high cost, and interpersonal variations. Although the manufacture of universal CAR-T (U-CAR-T) cells have significantly improved, they are still not a stable and unified cell bank. METHODS: Here, we tried to further improve the convenience and flexibility of U-CAR-T cells by constructing novel modular universal CAR-T (MU-CAR-T) cells. For this purpose, we initially screened healthy donors and cultured their T cells to obtain a higher proportion of stem cell-like memory T (TSCM) cells, which exhibit robust self-renewal capacity, sustainability and cytotoxicity. To reduce the alloreactivity, the T cells were further edited by double knockout of the T cell receptor (TCR) and class I human leukocyte antigen (HLA-I) genes utilizing the CRISPR/Cas9 system. The well-growing and genetically stable universal cells carrying the CAR-moiety were then stored as a stable and unified cell bank. Subsequently, the SDcatcher/GVoptiTag system, which generate an isopeptide bond, was used to covalently connect the purified scFvs of antibody targeting different antigens to the recovered CAR-T cells. RESULTS: The resulting CAR-T cells can perform different functions by specifically targeting various cells, such as the eradication of human immunodeficiency virus type 1 (HIV-1)-latenly-infected cells or elimination of T lymphoma cells, with similar efficiency as the traditional CAR-T cells did. CONCLUSION: Taken together, our strategy allows the production of CAR-T cells more modularization, and makes the quality control and pharmaceutic manufacture of CAR-T cells more feasible.

The receptor binding domain of SARS-CoV-2 Omicron subvariants targets Siglec-9 to decrease its immunogenicity by preventing macrophage phagocytosis.

The development of a vaccine specific to severe acute respiratory syndrome coronavirus 2 Omicron has been hampered due to its low immunogenicity. Here, using reverse mutagenesis, we found that a phenylalanine-to-serine mutation at position 375 (F375S) in the spike protein of Omicron to revert it to the sequence found in Delta and other ancestral strains significantly enhanced the immunogenicity of Omicron vaccines. Sequence FAPFFAF at position 371-377 in Omicron spike had a potent inhibitory effect on macrophage uptake of receptor-binding domain (RBD) nanoparticles or spike-pseudovirus particles containing this sequence. Omicron RBD enhanced binding to Siglec-9 on macrophages to impair phagocytosis and antigen presentation and promote immune evasion, which could be abrogated by the F375S mutation. A bivalent F375S Omicron RBD and Delta-RBD nanoparticle vaccine elicited potent and broad nAbs in mice, rabbits and rhesus macaques. Our research suggested that manipulation of the Siglec-9 pathway could be a promising approach to enhance vaccine response.