Bin Yu

Engineered extracellular vesicles (EVs) are promising therapeutic carriers for cancer treatment, but preserving intrinsic therapeutic activity while incorporating multiple immunomodulatory functions remains a challenge. Here, we developed a modular engineering strategy that combines the genetic engineering of EV-producing cells with bioorthogonal click chemistry to construct multifunctional oncolytic extracellular vesicles (OEVs). Azide-functionalized OEVs were generated from CD40L-expressing cells infected with a capsid-engineered oncolytic adenovirus. A toll-like receptor 9 (TLR9) agonist was subsequently conjugated to the OEV surface through click chemistry, integrating immune stimulation while retaining the oncolytic activity. The engineered OEVs efficiently infected tumor cells, induced antigen-presenting cell activation, enhanced intratumoral CD8+ T-cell responses, promoted B-cell activation and tumor-specific antibody production, and improved the therapeutic efficacy of the immune checkpoint blockade. These findings demonstrated that combining genetic engineering with bioorthogonal chemistry provided a strategy for constructing multifunctional oncolytic EVs with coordinated cytotoxic and immunomodulatory activities, offering an approach to next-generation cancer immunotherapies.

Oncolytic viruses have emerged as a promising strategy in cancer immunotherapy and have been applied to the treatment of various tumors. We previously demonstrated that oncolytic adenoviruses engineered expressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) could induce apoptosis in tumor cells and modulate immune responses in the tumor microenvironment. To enhance targeting capability and antitumor immunity, the full-length TRAIL is intended to be replaced with a soluble TRAIL (sTRAIL). However, the sTRAIL is limited by structural instability and hepatic accumulation. In this study, we performed an optimized design based on the previously developed, more stable sTRAIL (FA1FT) by fusing it with a knobless fiber motif derived from fowl adenovirus type 1. Through structural analysis and genetic engineering, we developed a novel recombinant sTRAIL protein (mut-FA1TLST) that exhibited reduced liver tropism and enhanced antitumor activity both in vitro and in vivo. Furthermore, we incorporated this optimized sTRAIL into a capsid-modified oncolytic adenovirus, which demonstrated improved tumor targeting ability and antitumor efficacy. The recombinant oncolytic virus expressing the sTRAIL also promoted activation of CD4⁺ T cells, CD8⁺ T cells and DCs in the tumor microenvironment, thereby eliciting antitumor immune response. Overall, this study provides a promising recombinant sTRAIL candidate for preclinical and clinical applications and presents a novel oncolytic virus with potential for in vivo tumor immunotherapy.

The primary aim of COVID-19 vaccine development is to induce highly efficient broadly neutralizing antibodies (bNAbs) against circulating and emergent SARS-CoV-2 variants. Rapid and sustained germinal center (GC) responses at an early stage are crucial to produce bNAbs. However, the mechanisms underlying the formation of early GC responses and strategies to effectively promote these responses remain to be further investigated. In this study, we found that the combination of anti-CD25 monoclonal antibodies (mAb) with the COVID-19 subunit vaccine significantly enhances cross-reactive neutralizing antibody responses in mice. Modulation of CD25 at different time points before and after vaccination resulted in varying effects on the GC response, with day 0 being the most effective in assisting the vaccine to induce a stronger GC response. This enhancement is achieved by rapidly inhibiting regulatory T (Treg) cells in draining lymph nodes, an effect observed not only in antigen-specific subsets but also across the bulk lymphocyte population—thereby creating a pro-immune microenvironment that facilitates the induction of an effective early GC response. This leads to the generation of more antigen-recognizing B cells and significantly increases both the potency and breadth of neutralizing antibody responses. Our findings propose a strategy to enhance vaccine efficacy against SARS-CoV-2 and other hypervariable pathogens by effectively promoting the development of early and robust GC responses.

Emerging viruses such as SARS-CoV-2 and HIV-1 pose major challenges for traditional vaccines due to their rapid mutation and immune evasion. Here, we present a modular liposomal nanoparticle (LNP) vaccine platform integrating trimeric immunogens and synergistic adjuvants to induce broad and durable immunity. Using a self-assembled SARS-CoV-2 RBD trimer (RM) based on MTQ and Ni²⁺/His-tag coupling, antigens were directionally displayed on LNPs coloaded with QS-21, MPLA, and R848 adjuvants. This RM-LNP formulation enhanced antigen stability, lymph node targeting, and germinal center responses, eliciting high titers of broadly neutralizing antibodies and strong T and memory B cell immunity. The RM-LNP vaccine conferred potent neutralization against Omicron subvariants and protected hACE2 mice from Delta, BA.5, and XBB infection. Extension to HIV-1 vaccine design also demonstrated significant and broad neutralization against Tier 2 strains. This study offers a versatile nanovaccine strategy for combating highly mutable viruses.

Viral infection generally induces polyclonal neutralizing antibody responses. However, how many lineages of antibody responses can fully represent the neutralization activities in sera has not been well studied. Using the newly designed stable HIV-1 Env trimer as hook, we isolated two distinct broadly neutralizing antibodies (bnAbs) from Chinese rhesus macaques infected with SHIV_1157ipd3N4 for 5 years. One lineage of neutralizing antibodies (JT15 and JT16) targeted the V2-apex in the Env trimers, similar to the J038 lineage bnAbs identified in our previous study. The other lineage neutralizing antibody (JT18) targeted the V3 crown region in the Env, which strongly competed with human 447-52D. Each lineage antibody neutralized a different set of viruses. Interestingly, when the two neutralizing antibodies from different lineages isolated from the same macaque were combined, the mixture had a neutralization breath very similar to that from the cognate sera. Our study demonstrated that a minimum of two different neutralizing antibodies can fully recapitulate the serum neutralization breadth. This observation can have important implications in AIDS vaccine design.

Enhancing the antitumor immune response and targeting ability of oncolytic viruses will improve the effect of tumor immunotherapy. Through infecting neural stem cells (NSCs) with a capsid dual-modified oncolytic adenovirus (CRAd), we obtained and characterized the "oncolytic extracellular vesicles" (CRAdEV) with improved targeted infection and tumor killing activity compared with CRAd. Both ex vivo and in vivo studies revealed that CRAdEV activated innate immune cells and importantly enhanced the immunomodulatory effect compared to CRAd. We found that CRAdEV effectively increased the number of DCs and activated CD4(+) and CD8(+) T cells, significantly increased the number and activation of B cells, and produced higher levels of tumor-specific antibodies, thus eliciting enhanced antitumor activity compared with CRAd in a B16 xenograft immunocompetent mice model. This study provides a novel approach to oncolytic adenovirus modification and demonstrates the potential of "oncolytic extracellular vesicles" in antitumor immunotherapy.

One of the major functions of the accessory protein Vif of human immunodeficiency virus type 1 (HIV-1) is to induce the degradation of APOBEC3 (A3) family proteins by recruiting a Cullin5-ElonginB/C-CBFβ E3 ubiquitin ligase complex to facilitate viral replication. Therefore, the interactions between Vif and the E3 complex proteins are promising targets for the development of novel anti-HIV-1 drugs. Here, peptides are designed for the Vif-CBFβ interaction based on the sequences of Vif mutants with higher affinity for CBFβ screened by a yeast surface display platform. We identified two peptides, VMP-63 and VMP-108, that could reduce the infectivity of HIV-1 produced from A3G-positive cells with IC50 values of 49.4 μM and 55.1 μM, respectively. They protected intracellular A3G from Vif-mediated degradation in HEK293T cells, consequently increasing A3G encapsulation into the progeny virions. The peptides could rapidly enter cells after addition to HEK293T cells and competitively inhibit the binding of Vif to CBFβ. Homology modeling analysis demonstrated the binding advantages of VMP-63 and VMP-108 with CBFβ over their corresponding wild-type peptides. However, only VMP-108 effectively restricted long-term HIV-1 replication and protected A3 functions in non-permissive T lymphocytes. Our findings suggest that competitive Vif-derived peptides targeting the Vif-CBFβ interaction are promising for the development of novel therapeutic strategies for acquired immune deficiency syndrome.

An oncolytic virus is a promising strategy for glioblastoma (GBM) therapy. However, there are still some challenges such as the blood-brain barrier (BBB) and preexisting immunity for targeted treatment of GBM with an oncolytic virus. In this study, two kinds of cell membrane-coated oncolytic adenoviruses (NCM-Ad and GCM-Ad) were prepared using neural stem cells (NSCs) and GBM cells as sources of membranes, respectively, and were shown to improve the targeted infectivity on GBM cells and avoid the immune clearance of preexisting neutralizing antibodies in vitro and in vivo. Specifically, NCM-Ad showed a strong ability to cross the BBB and target tumor cells in vivo. To improve the cytotoxicity to GBM, a capsid dual-modified oncolytic adenovirus (A4/k37) was also encapsulated, and NCM-A4/k37 showed outstanding tumor targeting and inhibition capacity in an orthotopic xenograft tumor model of GBM upon intravenous administration. This study provides a promising oncolytic virus-based targeted therapeutic strategy for glioma.

Background: In addition to specifically inducing tumor cell apoptosis, recombinant tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has also been reported to influence the cancer immune microenvironment; however, its underlying effects and mechanisms remain unclear. Investigating the immunomodulatory effects and mechanisms of recombinant TRAIL in the tumor microenvironment (TME) may provide an important perspective and facilitate the exploration of novel TRAIL strategies for tumor therapy. Methods: Immunocompetent mice with different tumors were treated with three doses of recombinant TRAIL, and then the tumors were collected for immunological detection and mechanistic investigation. Methodological approaches include flow cytometry analysis and single-cell sequencing. Results: In an immunocompetent mouse model, recombinant soluble mouse TRAIL (smTRAIL) had dose-related immunomodulatory effects. The optimal dose of smTRAIL (2 mg/kg) activated innate immune cells and CD8⁺ T cells, whereas higher doses of smTRAIL (8 mg/kg) promoted the formation of a tumor-promoting immune microenvironment to counteract the apoptotic effects on tumor cells. The higher doses of smTRAIL treatment promoted M2-like macrophage recruitment and polarization and increased the production of protumor inflammatory cytokines, such as IL-10, which deepened the suppression of natural killer (NK) cells and CD8⁺ T cells in the tumor microenvironment. By constructing an HU-HSC-NPG.GM3 humanized immune system mouse model, we further verified the immunomodulatory effects induced by recombinant soluble human TRAIL (shTRAIL) and found that combinational administration of shTRAIL and trabectedin, a macrophage-targeting drug, could remodel the tumor immune microenvironment, further enhance antitumor immunity, and strikingly improve antitumor effects. Conclusion: Our results highlight the immunomodulatory role of recombinant TRAIL and suggest promising therapeutic strategies for clinical application. Graphical