LNP-IL-21-engineered PBMC induced HBV clearance in BPS persistence mice
Previously, we described an IL-21-based cellular therapy, in which splenocytes from BPS persistence mice were subjected to recombinant IL-21 and rHBsAg co-stimulation before infusion into BPS persistence mice, could induce HBV clearance in nearly half of recipient mice in a cellular immune response dependent manner [8]. Given the ability of LNP-IL-21 administration to induce potent anti-HBV humoral and cellular immune responses in vivo (Figure 2-6 ) and the possibility and operability of LNP-IL-21-based cellular therapy, PBMC, but not splenocytes, were collected from BPS persistence mice and co-stimulated with rHBsAg and LNP-IL-21or LNP before injection into treatment-naïve BPS persistence mice (Figure 7A ). Compared to mice receiving LNP/rHBsAg co-treated PBMC, those receiving LNP-IL-21/rHBsAg co-treated PBMC displayed accelerated decreases of serum HBsAg and HBeAg (Figure 7B and D ), accompanied by HBsAb seroconversion (Figure 7C ) and transient ALT elevations (Figure 7E ), and remained negative for serum HBV antigens by 4 w.p.i., indicating activation of humoral and cellular immune responses. Accordingly, clearance of serum HBV antigens following injection of LNP-IL-21/rHBsAg-co-stimulated PBMC correlated with disappearance of BPS replicons DNA and capsids in mouse livers (Figure 7G ), proving true clearance.
Discussion
In practice, successful functional cure, termed as loss of serum HBsAg with or without HBsAg appearance, could be achieved in a small fraction of CHB patients after antiviral treatment. However, hepatitis B may be reactivated due to cccDNA persistence in hepatocytes [2, 6]. In an effort to achieve CHB pure cure that is removal of cccDNA from HBV infected hepatocytes, new therapeutic methods including drug candidates and delivery systems are urgently required [1, 6]. We previously reported that injection of IL-21-expressing AAV (AAV-IL-21) into both BPS-based and rcccDNA-based HBV persistence mice could induce clearance of serum HBV markers, and more importantly BPS replicons DNA and rcccDNA in liver tissues, via activation of HBV specific cytotoxic CD8+ T lymphocytes mediating removal of HBV-infected cells [7, 8]. Although AAV vectors have been approved for gene therapy in cancers and other diseases, an increasing body of evidence suggests AAV delivery system causes some adverse symptoms, such as abnormal immune response and DNA integration [10]. In the past few years, with technology advances in mRNA synthesis in vitro and delivery systems, LNP-based mRNA delivery has been applied broadly to clinical and basic research due to its acceptable safety profile [21, 24]. In this work, we compared gene expression profile of liver tissues between LNP-injected and AAV-injected mice via transcriptome sequencing. Although fewer DEGs were observed in LNP-injected mouse compared to AAV-injected mouse (Figure S3 ), the biofunctions of these DEGs are not fully understood and probably contribute to clarification of difference between these two systems. And, lipid and cholesterol, essential for maintenance of cell membrane structure, are the main components of LNP, suggesting LNP delivery system might be much safer than AAV. Next, we established LNP-IL-21particle and evaluated its therapeutic effects in animal models. In vitro study revealed that LNP-IL-21 displayed a well-formed spherical structure with diameter of 128.3 nm, and induced efficient IL-21 expression without obvious cytotoxicity in primary mouse and human hepatocytes (Figure 1, Figure S2 ). In vivo study indicated that livers in mice were the major organs targeted by LNP-IL-21, and were the main source of secreted IL-21 (Figure 1 ). Therefore, overall serum IL-21 levels were probably determined by half-time of IL-21 mRNA in hepatocytes and metabolic rate of serum IL-21. Meantime, storing LNP-IL-21 at 4℃ for 2 weeks did not affect its structure integrity and expression efficiency. In the following study, in order to further improve its tolerance to storage temperature and time, more works are needed to optimize LNP and IL-21 mRNA structure, such as modification of the proportion of each component in LNP and codon optimization of IL-21 open reading frame.
LNP-IL-21 treatment also achieved HBV pure clearance in BPS-based and rcccDNA-based HBV persistence mice (Figure 2-5 ) without provoking obvious changes in total bilirubin and creatine kinase (Figure S4-5 ), which is similarly observed in those AAV-IL-21-cured ones [8]. LNP-IL-21-incued clearance was associated with both cellular and anti-HBsAg humoral responses (Figure 6 ) while only cellular response was detected in AAV-IL-21-cured mice [8]. Previous studies revealed that IL-21 was involved in helping B cells to develop antibody-producing cells and in CD8+T cell activation [25, 26]. LNP-IL-21 treatment indeed improved the frequencies of HBsAg-specific B cells and viral specific T cell activities, contributing to HBV clearance (Figure 6 ). Given the facts that AAV itself is easy to establish persistent infection in humans [27] and that AAV-mediated HBV transduction in adult mice leads to HBV persistence [28], we speculated that AAV vector likely prohibited IL-21 from priming B cell activation during the process of AAV-IL-21-induced HBV clearance. IL-21 functions through binding to its receptor IL21R, broadly distributed on surface of different cells, to activate intracellular signaling pathways [25]. We previously observed that IL-21 could activate JAK-STAT pathway in CD8+ T cells in mice [8]. Future work delineating cellular and molecular mechanism for IL-21-mediated B and T cells activation in LNP-IL-21 treated mice would be carried out to identify key molecules and pathways through single-cell sequencing technology.
In order to avoid potential dangers caused by systemic administration of IL-21 with LNP-IL-21, we developed LNP-IL-21-based cellular therapy, in which PBMC were co-stimulated with LNP-IL-21 and rHBsAg before infusion into HBV persistence mice, and found it is much more sufficient in HBV clearance than IL-21-based cellular therapy [8], in which splenocytes were co-stimulated with recombinant IL-21 protein and rHBsAg. At the meantime, LNP-IL-21-based cellular therapy, but not IL-21-based cellular therapy, was able to activate both humoral and cellular responses. The possible reason is that IL-21 derived from the prokaryotic expression system in IL-21-based cellular therapy could not exert complete biological functions compared to IL-21 expressed from LNP-transfected cells in LNP-IL-21-based cellular therapy. Secondly, LNP-IL-21-based cellular therapy offers the possibility of autologous therapy for CHB patients. To achieve the goal, whether PBMC from patients could be switched from tolerant phase to activated state through LNP-IL-21-based cellular therapy treatment needs to be determined before infusion into patients. Lastly, in terms of mechanism research, ex vivo stimulated cells can be easily characterized, sorted and quantitated with regard to HBV specific humoral and cellular immune responses.