studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 461A
tion in HCC cell lines and enhanced the nuclear translocation
of TFEB-GFP and p62 clearance in all HCC cell lines. Western
blot analysis showed impaired expression of lysosomal
hydrolases cathepsin D in all HCC cell lines except HepG2
cells. Using real-time RT-PCR assay, we found that mRNA levels
of number of lysosomal genes (TFEB, cathepsin D) were significantly
reduced in all HCC cell lines compared to primary
human hepatocytes. Furthermore, we investigated whether
autophagy inhibitor (hydroxychloroquine) or inducer (Torin 1)
could be used as a therapeutic strategy for HCC. Interestingly,
long-term culture of HCC cells with autophagy inhibitor leads
to complete inhibition of HCC growth by MTT and cell colony
assay. Conclusion: Our results show that increased expression
of lysosomal efflux protein (p62) in HCC cell lines is due to
defects in mTOR-TFEB signaling. Our results also show that the
autophagy inhibitor hydroxychloroquine can be a potential
agent to inhibit HCC growth.
Disclosures:
The following authors have nothing to disclose: Rajesh Panigrahi, Partha K. Chandra,
Pauline Ferraris, Fatma Aboulnasr, Srinivas Chava, Tong Wu, Srikanta Dash
502
Chimeric hepatitis E virus-like particles as a tool of oral
vaccination against hepatitis A virus
Eun Byul Lee 1 , Jung-Hee Kim 1 , Jung Eun Choi 1 , Seung Kew
Yoon 1,2 ; 1 The Catholic University Liver Research Center & WHO
Collaborating Center of Viral Hepatitis, Seoul, Korea (the Republic
of); 2 College of Medicine, The Catholic University of Korea, Seoul,
Korea (the Republic of)
Background: Virus-like particles (VLPs) has been shown the possibility
of new tools for oral vaccination because VLPs can be
protected against harsh environment of the human digestive
tract. Recent studies have demonstrated that hepatitis E virus
(HEV)-LPs can induce both mucosal and systemic immunity after
oral administration. Currently, commercial vaccine against
hepatitis A virus (HAV) are in the intravenous form, but those
for oral administration are yet to be developed. In this study,
we investigated whether HEV-LPs containing HAV antigens
(HEV-LP-HAVag) could be useful delivery tool for oral vaccination
against HAV. Methods: To produce HEV-LPs from mammalian
cells, Huh7 cells were infected with the recombinant
baculovirus containing CMV promoter derived-Nt-ORF2 gene
(Bac-Nt-ORF2). Nt-ORF2 expression in Huh7 cells was confirmed
by western blot analysis, and HEV-LPs produced from
Huh7 cells were purified by sucrose gradient centrifugation.
The morphology of purified HEV-LPs was observed by electron
microscopy (EM). Expression vectors of HAV antigens (VP1,
VP3, VP1-P2A)-derived from HM175 strain were constructed
by insertion of gene encoding each HAV antigen into pcDNA3
vector in frame. To establish HEV-LPs packing system carrying
HAV antigen expression vectors, HEV-LPs were disassembled
using biochemical buffer containing DTT and low concentration
of CaCl 2,
and then HAV antigen expression vectors were
packed into the re-assembled HEV-LPs by increasing concentration
of CaCl 2.
Delivery of HAV antigen expression vectors and
their expression were confirmed in Huh7 cells infected HEV-
LP-HAVag by western blot analysis. Results: Nt-ORF2 was successfully
expressed in Huh7 cells infected with Bac-Nt-ORF2.
Moreover, Nt-ORF2 expressed in Huh7 cells formed HEV-LPs
of 25 nm in diameter through their self-assembly property. Furthermore,
HEV-LP-HAVags were efficiently build up by in vitro
disassembly/reassembly systems and, their morphology was
well preserved compared to that of empty HEV-LPs. By infection
with HEV-LP-HAVags, HAV antigens expression vectors were
delivered to Huh7 cells, and subsequently 3 kinds of HAV antigens
were efficiently expressed in the cells. Conclusion: We
established an antigen-encapsulation system with HEV-LPs and
successfully delivered the antigen expression vectors using the
system. This might be useful tool for development of vaccine as
well as vesicles for gene therapy.
Disclosures:
The following authors have nothing to disclose: Eun Byul Lee, Jung-Hee Kim, Jung
Eun Choi, Seung Kew Yoon
503
High-throughput sequencing to define the miRNA cargo
of LSEC-derived, anti-viral exosomes
Michael Kriss 1 , Michael Edwards 3 , Lucy Golden-Mason 1 , Colin
Larson 4 , Katrina Diener 4 , Hugo R. Rosen 1,2 ; 1 Division of Gastroenterology
& Hepatology, University of Colorado Anschutz Medical
Campus, Aurora, CO; 2 Denver VA Medical Center, Denver,
CO; 3 Division of Pulmonary Sciences and Critical Care Medicine,
University of Colorado Anschutz Medical Campus, Aurora, CO;
4 Genomics and Microarray Core, University of Colorado Anschutz
Medical Campus, Aurora, CO
Aim: To characterize the miRNA content of LSEC-derived exosomes
to identify novel anti-viral mediators. Methods: Exosomes
were isolated from supernatants of an immortalized LSEC cell
line cultured for 48h ± rhIFNα (100ng/ml) or ± IFNλ1/2/3
(100ng/ml each) using ExoQuick-TC (SBI). Total RNA was isolated
using SeraMir RNA extraction kit (SBI). Small RNA library
was generated using Illumina TruSeq Small RNA sample prep
kit (1μg input RNA) and validated on Agilent Bioanalyzer.
Sequencing was performed using Illmina HiSEQ 2500 Platform
using V4 Chemistry Single Read 1x50. The sequenced reads
were aligned with TopHat2 against both the human mirBASE
and hg38 databases separately, and quantitated using Partek
Genomics Suite v6.6. The average amount of reads produced
from the miRNA and genome alignments were approximately
3.3 and 9.2 million reads respectively per sample. Results:
Exosomes derived from LSECs stimulated with either rhIFNα
or IFNλ1/2/3 suppressed HCV replication in vitro and led
to induction of ISGs in HCV-infected hepatocytes, consistent
with prior reports. Electrophoresis of isolated RNA revealed
absence of ribosomal RNA (18s and 28s) compared to RNA
isolated from derivative cells. High throughput sequencing was
performed with a base call accuracy of 99.96% (Phred Q score
33.813±0.257). miRNA fold changes relative to mock were
calculated based on read counts for all targets with greater
than 100 total read counts. The top ten fold changes for each
treatment group are included in table 1. miR-3168 was the
only miRNA uniquely induced in the IFNλ-treatment group.
miR-122 was present in all samples with modest induction in
both IFNλ (1.31-fold) and IFNα (1.75-fold) treatment groups.
Additional alignment with hg38 database revealed presence
of miR-138 in only the IFNλ and IFNα treated groups. Conclusions:
LSEC-derived exosomes differentially regulate miRNA in
the setting of type I and type III interferons. This may contribute
to the anti-viral effect of these exosomes and provide novel
insights into therapeutic mechanisms moving forward.