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Generating a robust cytotoxic T-cell reaction is a crucial requirement for effective immune-based treatment against various viral infections and cancers. Nucleotide-based vaccines, including mRNA vaccines, have demonstrated their ability to serve as potent triggers for this cytotoxic immune response. Nucleotide vaccines, which encompass mRNA vaccines along with their capability for intracellular antigen production, have been demonstrated to effectively stimulate a robust cytotoxic immune response.
The precise mechanism by which mRNA vaccines are delivered intracelluarly to the cytosol of immune cells responsible for presenting antigens remains inadequately comprehended. In this report, they present their work on creating a lipid nanoparticle formation designed to transport mRNA vaccines, with the intention of triggering a cytotoxic CD8 T cell response.
They demonstrate the successful introduction of genetic material into dendritic cells, macrophages, and neutrophils. The effectiveness of the vaccine was evaluated in a rigorous B16F10 melanoma model. Following a single immunization, they observed a significant activation of CD8 T-cells. Treating B16F10 melanoma tumors with lipid nanoparticles containing mRNA encoding tumor-associated antigens gp100 and TRP2 resulted in tumor regression and prolonged survival in treated mice. The immune response can be further heightened by including the adjuvant LPS. To sum up, the lipid nanoparticle formulation introduced here holds promise as a delivery vehicle for mRNA vaccines, capable of provoking a robust cytotoxic T-cell response. Additional optimization, such as integrating diverse adjuvants, is likely to enhance the vaccine's effectiveness.
Cancer immunotherapy relies on the immune system's capacity to identify and eliminate cancerous cells. Recent trials in clinical settings, involving the evaluation of checkpoint inhibitors or the transfer of adoptive T cells, have demonstrated the ability of antigen-specific T cells to manage cancer.
In order to utilize the immune system for cancer treatment, it is essential to create approaches that counteract inflammation promoting tumor growth, alter the tumor's surrounding environment that influences T cell function, and enhance the diversity of T cell responses through vaccination. The adaptive immune system functions to safeguard us against recurrent infections through its dual components: the humoral arm, comprising antibodies, and the cellular arm, comprising T cells. Antibodies serve as valuable instruments for eliminating extracellular pathogens and toxins. Nevertheless, when it comes to specific intracellular pathogens and tumors, specialized T cells, referred to as cytotoxic T cells (CTLs) or cluster of differentiation 8 (CD8) T cells, are essential.
Nucleotide vaccines possess an appealing ability to stimulate a potent CD8 T cell response mediated by Major Histocompatibility Complex I (MHC-1). Nevertheless, effectively delivering them to target cells with minimal harm remains a challenge. Obstacles in the delivery of mRNA vaccines include: safeguarding mRNA from degradation by ubiquitous endonucleases, ensuring its arrival at target cells, and facilitating both endocytosis and escape from endosomes before degradation. Various approaches have been developed for the successful delivery of mRNA vaccines, including mRNA encapsulation within viral and nanoparticle carriers, as well as sequence optimization to enhance stability and tailor immunogenicity.
A collection of lipid nanoparticles(LNPs) has recently been engineered in this lab to facilitate mRNA delivery to hepatocytes. Numerous carriers for the intracellular transfer of oligonucleotides have been designed in different forms and dimensions. Nevertheless, nanoparticles within a size spectrum of approximately 200 nm appear to be especially suitable for facilitating the delivery of mRNA vaccines.
Specialized antigen-presenting cells(APCs), notably dendritic cells (DCs), hold significant significance in eliciting T cell immunity. These APCs are abundant within lymph nodes and consistently sample the surrounding interstitial fluid. For nanoparticles to effectively drain into the lymph nodes and successfully transfect APCs, factors such as nanoparticles size, charge, and colloidal stability play a crucial role.
In comparison to other delivery methods, lipid nanoparticles(LNPs) present several advantages, which include: (i) robust synthesis process allowing for flexible adjustment of components and composition to enhance delivery efficiency and reduce toxicity, (ii) the potential incorporation of immune enhancers like adjuvants or immune cell-targeting ligands to customize the immune response, and (iii) successful historical utilization of LNPs for mRNA vaccine delivery. Notably, the earliest LNP formulation for mRNA vaccine delivery can be traced back to Martinon et al. in 1993. Clinical experience with mRNA vaccines thus far has been highly positive: No significant adverse effects have been reported, and antigen-specific immune responses have been observed in some patients. As of now, we are only aware of one clinical study involving lipid nanoparticles as an mRNA carrier that has published results, along with one ongoing study.
This formulation is composed of various components, including an ionizable lipid, a phospholipid, cholesterol, a lipid-containing polyethylene glycol(PEG), and an additive intended for delivering mRNA vaccines. The ionizable lipid possesses a positive charge under low pH conditions, facilitating its binding with the negatively charged mRNA. This interaction may also contribute to cellular uptake and the escape of the complex from endosomes. Both the phospholipid and cholesterol play vital roles in maintaining the stability of the lipid nanoparticles(LNPs) and could potentially aid in endosomal escape. The incorporation of PEGylated lipids serves to prevent aggregation of LNPs, assists in their distribution throughout the body, and reduces non-specific interactions.
The authors' hypothesis centers on the possibility of enhancing the delivery of mRNA vaccines through the optimization of LNPs composed of these elements, ultimately leading to the induction of a robust CD8 T cell immune response. Regarding the assessment of the potential expansion of antigen-specific T cell populations, a suitable in vitro assay is lacking. This is due to the fact that both T cell counts and antibody titers are influenced by multifaceted factors beyond the mere transfection efficiency of a specific immune cell type. They are also contingent on the intricate immunological signaling cascade required for an effective immune response to occur. Consequently, a comprehensive range of LNP formulations was fine-tuned to provoke a potent T-cell response in an in vivo context.
The author formulated and fine-tuned a collection of LNPs, each carrying mRNA encoding the model immunological protein ovalbumin(OVA). To evaluate their effectiveness, each variant was administered subcutaneously into groups of five C57BL/6 mice, injecting 10ug of mRNA per mouse into the lower back(dorsal posterior) area. Seven days after a single injection, mice were subjected to blood collection. Subsequently, the red blood cells were lysed, and the monocytes were labeled using a tetramer conjugate specific for the OVA-epitope SIINFEKL. This process aimed to ascertain the proportion of OVA-specific CD8 T cells.
