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.