Jianzhu Chen

Messenger RNA (mRNA) vaccines have emerged as a powerful vaccination platform, yet efficient and targeted delivery of mRNA to professional antigen-presenting cells (APCs) remains a major challenge. Here, we developed a dendritic cell (DC)-targeting virus-like particle (DVLP) platform for mRNA delivery by combining human immunodeficiency virus (HIV) Gag fused with the bacteriophage MS2 coat protein, which recognizes mRNA bearing MS2 stem-loop sequences and an engineered Sindbis virus glycoprotein mutant (SV-Gmt) that preferentially targets dendritic cells. African swine fever virus (ASFV) P30 was employed as a model antigen to evaluate platform performance. In vitro , SV-Gmt-pseudotyped VLPs encapsulating P30 mRNA (DVLP-P30) preferentially delivered antigen mRNA to dendritic cells compared with broad-tropic vesicular stomatitis virus G protein (VSV-G)-pseudotyped VLPs encapsulating P30 mRNA (VSV-G VLP-P30). In mice, vaccination with DVLP-P30 induced significantly higher P30-specific antibody responses than VSV-G VLP-P30. Enhanced humoral immunity was accompanied by increased frequencies of CD4 ⁺ and CD8 ⁺ effector memory T cells, indicating improved generation of antigen-specific immune memory. Notably, DC-targeted mRNA delivery promoted stronger interferon-γ responses and increased IgG2a production, consistent with a Type 1 helper T cell (Th1)-skewed immune profile. Together, these findings demonstrate that DC-targeting VLPs function as effective mRNA carriers and enhance both the magnitude and quality of vaccine-induced immunity. This work establishes a versatile platform for targeted mRNA delivery and supports further development of DVLPs for prophylactic and therapeutic vaccination.

Studying antibody-mediated inhibition of ligand-receptor interactions is critical for understanding fundamental biological processes and developing therapeutic interventions. However, for high-consequence viruses with entry machinery involving multiple viral proteins, it is often difficult to develop pseudotyped viruses that can infect susceptible host cells. Here, we report a rapid platform for detecting antibody-mediated inhibition of ligand-receptor interactions by decoupling viral attachment from post-entry membrane fusion and gene expression. Our platform leverages HIV Gag-based self-assembling virus-like particles (VLPs) where Gag is fused with GFP and further pseudotyped with selected ligands for easy readout of VLP binding to susceptible cells. We first validated the platform’s stringent cell-type specificity using broadly tropic VSV-G and CD19-specific single chain fragment variable (scFv) pseudotypes. Next, we applied this technology to African swine fever virus (ASFV) - a high-consequence agricultural pathogen lacking surrogate neutralization assays due to its multi-protein entry machinery. By screening VLPs pseudotyped with different viral proteins against diverse mammalian cell lines, we found that VLP pseudotyped with ASFV CD2v binds specifically to Vero cells. Leveraging this interaction, we developed a rapid test for detecting antibodies that inhibit CD2v-pseudotyped VLP binding to Vero cells. Longitudinal analysis of sera from mRNA-LNP vaccinated pigs showed that the degrees of VLP-based binding-inhibition strongly correlated with the levels of anti-CD2v IgG, validating the assay’s utility. Overall, the pseudotyped VLP platform provides an enabling tool for ligand-receptor discovery, vaccine evaluation, and disease surveillance for select agents.

Solid tumors evade immunotherapy because of immunosuppressive microenvironments that limit immune cell persistence and function. Although interleukin-12 (IL-12) potently activates antitumor immunity, its clinical use has been constrained by systemic toxicity. Here we engineered a CAR natural killer (NK) cell platform that integrated IL-12 signaling to enhance antitumor immunity. IL-12 signaling synergized with CAR activation to sustain mTORC1 activity through convergent Ras-ERK and PI3K pathways, promoting metabolic fitness, autonomous expansion, and sustained effector function. IL-12 also activated bystander NK cells, T cells, and macrophages to remodel the tumor microenvironment. To improve safety, IL-12 was tethered to a collagen-binding A3-domain, restricting its activity to the extracellular matrix and limiting systemic exposure. In ovarian and pancreatic cancer models, matrix-anchored IL-12 CAR NK cells expanded without exogenous cytokines and achieved durable tumor control. These findings suggest that mTORC1-mediated integration of CAR and cytokine signaling can enhance immune fitness and overcome immunosuppressive tumor microenvironments.

Background/Objective: Persistent infection with human papillomavirus type 16 (HPV-16) is the principal cause of cervical high-grade squamous intraepithelial lesions (cHSIL) and cervical cancer, yet the established treatments remain limited to ablative or excisional procedures that carry reproductive risk and do not eliminate the underlying infection. We report the preclinical development of ARV-2001, a messenger RNA (mRNA)–lipid nanoparticle (LNP) immunotherapeutic encoding mutated, non-oncogenic HPV-16 E6 and E7 fused to a SARS-CoV-2 spike S2 subdomain enriched in human CD4 helper epitopes, formulated in a novel cholesterol-derived ionizable lipid (ARV-T1). Methods: Interactions of ARV-2001-expressed antigens with p53 and retinoblastoma (Rb) were evaluated in human cervical carcinoma cell line C33A, in lentiviral constructs in primary human keratinocytes, and in soft-agar colony-formation assays. ARV-2001 was administrated by intramuscular (IM) or intradermal (ID) injection in naive mice or in the TC-1 tumor models. Tumor size and survival were monitored over time and tumor-infiltrated lymphocytes were characterized by flow cytometry. Intracellular cytokine staining and Elispot were used to evaluate immunogenicity. Results: In vitro, the mutated E6/E7–S2 antigen lost the ability to degrade p53, to deregulate the retinoblastoma (Rb) pathway, and to support anchorage-independent growth, suggesting abrogation of oncogenic activity. The S2 domain and imiquimod administration each augmented antitumor activity and intratumoral CD8⁺ T-cell infiltration while reducing myeloid-derived suppressor cells in the syngeneic HPV-16 E6/E7 TC-1 tumor models. In addition, ID administration of ARV-2001 into TC-1 tumor-bearing mice was superior to IM administration in terms of both tumor growth inhibition and survival. ID vaccination with ARV-2001 in mice consistently elicited a more potent E6/E7-specific T-cell response than the same dose given IM. Dose-escalation studies showed a dose-dependent T cell response against E6/E7 in ID-injected mice. Conclusions: This study supports future human evaluation of intradermally administrated ARV-2001 for treatment of HPV-16+ cHSIL in clinical trials.

Allogeneic cellular immunotherapy exhibits promising efficacy for cancer treatment, but donor cell rejection remains a major barrier. Here, we systematically evaluate human leukocyte antigens (HLA) and immune checkpoints PD-L1, HLA-E, and CD47 in the rejection of allogeneic NK cells and identify CD8⁺ T cells as the dominant cell type mediating allorejection. We demonstrate that a single gene construct that combines an shRNA that selectively interferes with HLA class I but not HLA-E expression, a chimeric antigen receptor (CAR), and PD-L1 or single-chain HLA-E (SCE) enables the one-step construction of allogeneic CAR-NK cells that evade host-mediated rejection both in vitro and in a xenograft mouse model. Furthermore, CAR-NK cells overexpressing PD-L1 or SCE effectively kill tumor cells through the upregulation of cytotoxic genes and reduced exhaustion and exhibit a favorable safety profile due to the decreased production of inflammatory cytokines involved in cytokine release syndrome. Thus, our approach represents a promising strategy in enabling “off-the-shelf” allogeneic cellular immunotherapies.

Viral capsid proteins are widely explored for subunit vaccine development but are often hampered by their complexity of production and low immunogenicity. Here, we report a simple approach to overcoming these challenges by combining mRNA vaccine technology with protein engineering. Using African swine fever virus (ASFV) capsid proteins P72 and penton as models, we engineered them into membrane-bound and secreted forms and compared their immunogenicity to that of the native intracellular form in mice and pigs through mRNA vaccination. The membrane-bound and secreted P72 and penton folded into their native multimeric structure independent of the viral chaperone, therefore preserving their conformational epitopes. The membrane-bound P72 and penton also elicited significantly stronger antibody and T cell responses than their secreted or intracellular counterparts. Our study provides a simple approach to enhancing folding, multimeric structure formation, and immunogenicity of viral capsid proteins for ASFV subunit vaccine development and immunogenicity of intracellular proteins in general.

Development of safe and effective subunit vaccines for controlling African Swine Fever Virus (ASFV) infection has been hampered by a lack of protective viral antigens, complex virion structures, and multiple mechanisms of infection. Here, we selected ASFV antigens based on their localization on the virion, known functions, and homologies to the subunits of the protective vaccinia virus vaccine. We also engineered viral capsid proteins for inducing optimal antibody responses and designed T cell-directed antigen for inducing broad and robust cellular immunity. The selected antigens in lipid nanoparticle-mRNA formulations were evaluated for immunogenicity in both mice and pigs with concordant results. Different antigens induced divergent immune response profiles, including the levels of IgG and T cell responses and effector functions of anti-sera. We further developed a computational approach to combine antigens into cocktails for inducing specific immune response profiles and validated candidate cocktail vaccines in mice. Our results provide a basis for further evaluating candidate subunit mRNA vaccines in challenge studies. Teaser Novel strategies to develop subunit vaccines for ASFV and other complex large DNA viruses.

Viral capsid proteins are widely explored for subunit vaccine development but are often hampered by their complexity of production and low immunogenicity. Here, we report a simple approach to overcome these challenges by combining mRNA vaccine technology with protein engineering. Using African swine fever virus (ASFV) capsid proteins P72 and penton as models, we engineered them into membrane-bound and secreted forms, and compared their immunogenicity to the native intracellular form in mice and pigs through mRNA vaccination. The membrane-bound and secreted P72 and penton folded into their native multimeric structure independent of viral chaperone, therefore preserving their conformational epitopes. The membrane-bound P72 and penton also elicited significantly stronger antibody and T cell responses than their secreted or intracellular counterparts. Our study provides a simple approach to enhance folding, multimeric structure formation, and immunogenicity of viral capsid proteins for ASFV subunit vaccine development and immunogenicity of intracellular proteins in general.