Evaluating Anti-tumor Immune Responses of Protein Nanoparticle-Based Cancer Vaccines.

Cancer vaccines displaying tumor-associated antigens (TAAs) train the immune system for enhanced tumor recognition and elimination. Nanoparticle-based cancer vaccines are ingested and processed by dendritic cells, which subsequently activate antigen-specific cytotoxic T cells, allowing them to identify and eliminate tumor cells displaying these TAAs. Here, we describe the procedures to conjugate TAA and adjuvant to a model protein nanoparticle platform (E2), followed by assessment of vaccine performance. Utilizing a syngeneic tumor model, the efficacy of in vivo immunization was determined by cytotoxic T lymphocyte assays and IFN-γ ELISpot ex vivo assays to measure tumor cell lysis and TAA-specific activation, respectively. In vivo tumor challenge directly allows evaluation of anti-tumor response and survival over time.

Nanoparticle vaccines can be designed to induce pDC support of mDCs for increased antigen display.

Cancer vaccine immunotherapy facilitates the immune system's recognition of tumor-associated antigens, and the biomolecular design of these vaccines using nanoparticles is one important approach towards obtaining strong anti-tumor responses. Following activation of dendritic cells (DCs), a robust CD8+ T cell-mediated adaptive immune response is critical for tumor elimination. While the role of efficient antigen-presenting myeloid DCs (mDCs) is conventionally attributed towards vaccine efficacy, participation by highly cytokine-producing plasmacytoid DCs (pDCs) is less understood and is often overlooked. We examined vaccines based on the E2 protein nanoparticle platform that delivered encapsulated TLR9 agonist bacterial-like DNA (CpG1826 or CpG1018) or TLR7 agonist viral ssRNA to determine their efficacy over free agonists in activating both mDCs and pDCs for antigen presentation. Although mDCs were only activated by nanoparticle-encapsulated TLR9 agonists, pDCs were activated by all the individually tested constructs, and CpG1826 was shown to induce pDC cytokine production. Transfer of secreted factors from pDCs that were stimulated with a vaccine formulation comprising peptide antigen and CpG1826 enhanced mDC display of the antigen, particularly when delivered in nanoparticles. Only when treated with nanoparticle-conjugated vaccine could pDCs secrete factors to induce antigen display on naïve mDCs. These results reveal that pDCs can aid mDCs, highlighting the importance of activating both pDCs and mDCs in designing effective cancer vaccines, and demonstrate the advantage of using nanoparticle-based vaccine delivery.

Macromolecular assembly of bioluminescent protein nanoparticles for enhanced imaging.

Bioluminescence imaging has advantages over fluorescence imaging, such as minimal photobleaching and autofluorescence, and greater signal-to-noise ratios in many complex environments. Although significant achievements have been made in luciferase engineering for generating bright and stable reporters, the full capability of luciferases for nanoparticle tracking has not been comprehensively examined. In biocatalysis, enhanced enzyme performance after immobilization on nanoparticles has been reported. Thus, we hypothesized that by assembling luciferases onto a nanoparticle, the resulting complex could lead to substantially improved imaging properties. Using a modular bioconjugation strategy, we attached NanoLuc (NLuc) or Akaluc bioluminescent proteins to a protein nanoparticle platform (E2), yielding nanoparticles NLuc-E2 and Akaluc-E2, both with diameters of ∼45 â€‹nm. Although no significant differences were observed between different conditions involving Akaluc and Akaluc-E2, free NLuc at pH 5.0 showed significantly lower emission values than free NLuc at pH 7.4. Interestingly, NLuc immobilization on E2 nanoparticles (NLuc-E2) emitted increased luminescence at pH 7.4, and at pH 5.0 showed over two orders of magnitude (>200-fold) higher luminescence (than free NLuc), expanding the potential for imaging detection using the nanoparticle even upon endocytic uptake. After uptake by macrophages, the resulting luminescence with NLuc-E2 nanoparticles was up to 7-fold higher than with free NLuc at 48 â€‹h. Cells incubated with NLuc-E2 could also be imaged using live bioluminescence microscopy. Finally, biodistribution of nanoparticles into lymph nodes was detected through imaging using NLuc-E2, but not with conventionally-labeled fluorescent E2. Our data demonstrate that NLuc-bound nanoparticles have advantageous properties that can be utilized in applications ranging from single-cell imaging to in vivo biodistribution.

Advancements in protein nanoparticle vaccine platforms to combat infectious disease.

Infectious diseases are a major threat to global human health, yet prophylactic treatment options can be limited, as safe and efficacious vaccines exist only for a fraction of all diseases. Notably, devastating diseases such as acquired immunodeficiency syndrome (AIDS) and coronavirus disease of 2019 (COVID-19) currently do not have vaccine therapies. Conventional vaccine platforms, such as live attenuated vaccines and whole inactivated vaccines, can be difficult to manufacture, may cause severe side effects, and can potentially induce severe infection. Subunit vaccines carry far fewer safety concerns due to their inability to cause vaccine-based infections. The applicability of protein nanoparticles (NPs) as vaccine scaffolds is promising to prevent infectious diseases, and they have been explored for a number of viral, bacterial, fungal, and parasitic diseases. Many types of protein NPs exist, including self-assembling NPs, bacteriophage-derived NPs, plant virus-derived NPs, and human virus-based vectors, and these particular categories will be covered in this review. These vaccines can elicit strong humoral and cellular immune responses against specific pathogens, as well as provide protection against infection in a number of animal models. Furthermore, published clinical trials demonstrate the promise of applying these NP vaccine platforms, which include bacteriophage-derived NPs, in addition to multiple viral vectors that are currently used in the clinic. The continued investigations of protein NP vaccine platforms are critical to generate safer alternatives to current vaccines, advance vaccines for diseases that currently lack effective prophylactic therapies, and prepare for the rapid development of new vaccines against emerging infectious diseases. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Biology-Inspired Nanomaterials > Protein and Virus-Based Structures.

Mouse Models for Immunotherapy in Hepatocellular Carcinoma.

Liver cancer is one of the dominant causes of cancer-related mortality, and the survival rate of liver cancer is among the lowest for all cancers. Immunotherapy for hepatocellular carcinoma (HCC) has yielded some encouraging results, but the percentage of patients responding to single-agent therapies remains low. Therefore, potential directions for improved immunotherapies include identifying new immune targets and checkpoints and customizing treatment procedures for individual patients. The development of combination therapies for HCC is also crucial and urgent and, thus, further studies are required. Mice have been utilized in immunotherapy research due to several advantages, for example, being low in cost, having high success rates for inducing tumor growth, and so on. Moreover, immune-competent mice are used in immunotherapy research to clarify the role that the immune system plays in cancer growth. In this review paper, the advantages and disadvantages of mouse models for immunotherapy, the equipment that are used for monitoring HCC, and the cell strains used for inducing HCC are reviewed.