CN108795882B

CN108795882B - Methods for modulating hepatitis E virus assembly and stability of capsid protein ORF2

Info

Publication number: CN108795882B
Application number: CN201810580468.0A
Authority: CN
Inventors: 王佑春; 许楠; 黄维金; 赵晨燕; 张黎
Original assignee: National Institutes for Food and Drug Control
Current assignee: National Institutes for Food and Drug Control
Priority date: 2018-06-07
Filing date: 2018-06-07
Publication date: 2022-07-08
Anticipated expiration: 2038-06-07
Also published as: CN108795882A

Abstract

The present invention relates to methods of improving hepatitis e virus production, HEV virus replication, viral ORF2 protein stability, and HEV virus-like particle formation by HDAC6 inhibitors. The invention also relates to cell culture systems, and cell cultures for the production of hepatitis E virus, virus-like particles, and ORF2 protein. Furthermore, the present invention also relates to methods of treating HEV viral infection by increasing expression and/or activity of HDAC6in a cell.

Description

Methods for modulating hepatitis E virus assembly and stability of capsid protein ORF2

Technical Field

The present invention relates to the fields of molecular biology and virology. In particular, the invention relates to acetylated Hepatitis E Virus (HEV) capsid protein ORF2, and methods of modulating the level of acetylation of a Hepatitis E virus ORF2 polypeptide. The invention also relates to the use of inhibitors of HDAC6 for improving ORF2 polypeptide stability, HEV virus formation, HEV virus replication, and HEV virus-like particle formation. The invention also relates to methods of treating HEV viral infection by altering the acetylation level of HEV ORF2 in a cell.

Background

Hepatitis E Virus (HEV), which infects humans and animals worldwide, causes enterotransmitted viral hepatitis with a fatality rate of 25% in pregnant women. In 1989, hepatitis E virus sequences from enterically transmitted non-A, non-B hepatitis patients were reported, similar to sequences isolated from pigs, rabbits, rats, deer and wild boars. The capsid protein ORF2 of HEV is the main antigen of the virus, specifically binds to the 5' end of HEV genome RNA, and plays an important role in viral infection and assembly and maintenance of viral genome integrity. However, little is currently known about the regulation of ORF2 polypeptide stability and viral Inclusion Body (IB) formation in HEV infected cells.

Hepatitis E Virus (HEV), a non-enveloped virus, contains a 7.2kb single-stranded positive-strand RNA encoding the three proteins ORF1, ORF2 and ORF3 (1). There are four major HEV genotypes that cause approximately 2000 million human infections annually worldwide, including 3.3 million symptomatic hepatitis e cases and 56600 hepatitis e deaths (2, 3).

HEV genotypes

1 and 2 primarily infect humans and cause epidemic outbreaks in developing countries, and genotypes 3 and 4 have zoonosis in developing and developed countries.

ORF2 is the major antigen of HEV, comprising 660 amino acid residues, and plays a major role in viral infection, inducing a host immune response (5). The truncated ORF2(aa112-660) fragment was able to self-assemble into virus-like particles (VLPs) (6). The ORF2 VLPs can induce the same immunogenic pathway as the native virus, and have been used to mimic HEV in research and vaccine production (7, 8). At the same time, HEV ORF2 is also capable of binding to the 5' end of HEV genomic RNA, playing a role in viral assembly and maintenance of genomic integrity (9). The N-terminal signal sequence of ORF2 is co-translated in the Endoplasmic Reticulum (ER) and N-linked glycosylation modifications are achieved in the ER (10). In addition, the C-terminal 52 amino acid residues of ORF2 are also involved in HEV packaging and stabilization of capsid particles (11). Although ORF2 is very important in the HEV life cycle, it is unclear how it is regulated in the host.

Lysine acetylation is an important post-translational modification (PTM), dynamically catalyzed by lysine acetyltransferase, and removed by lysine deacetylase. Acetylation of lysine is involved in a variety of key events in the viral life cycle, including chromatin remodeling, gene expression and protein stability regulation, intracellular trafficking and lytic-latent transformation of the virus (13). HDAC6 is a member of the HDAC enzyme family of class IIb, deacetylating non-histones such as alpha-tubulin, HSP90 and p300 (14-17). HDAC6 plays an important role in the regulation of cell morphology, adhesion and migration, degradation and stress responses of misfolded proteins, tumor cell invasion, angiogenesis, and drug resistance (18-30). Recent studies have shown that HDAC6 also plays an important role in viral infection and pathogenesis (13). However, HDAC6 appears to have a dual role (13). In one aspect, HDAC6 is involved in host defense against viral infection (31-35). On the other hand, HDAC6 is utilized by viruses to help the viruses enter host cells or viral RNA synthesis (36-38). To date, only the HIV-1Tat protein in the virus has been identified as a HDAC6 substrate (35).

In view of the present serious health risks to humans from hepatitis e and the rapidly increasing trend in incidence in recent years, there is still a need for new methods for improving HEV virus vaccine production and treating HEV virus infection. Through intensive studies, the present inventors found that HEV utilizes a host's post-translational modification system to maintain HEV capsid protein ORF2 stability and a new mechanism of Inclusion Body (IB) formation. Based on this finding, the present inventors propose novel methods and compositions for improving HEV vaccine production and treating HEV infection by targeted modulation of the acetylation level of ORF2 in host cells.

Summary of The Invention

Accordingly, in one aspect, the invention provides a method and cell culture system for improving the production of HEV virus, virus-like particle (VLP) or capsid protein ORF2, comprising modulating the level of acetylation at position K411 of HEV capsid protein ORF2 in a producer cell. In one embodiment, an HEV virus is produced. In a further embodiment, the producer cells are mammalian cells, preferably A549 cells and PLC/PRF/5 cells, that can be used for the in vitro culture and production of HEV viruses. In a further embodiment, the method comprises the steps of: infecting the cells with HEV virus. In another embodiment, an HEV virus-like particle (VLP) or ORF2 polypeptide is produced. In further embodiments, the producer cell is a cell comprising a nucleic acid encoding ORF2, such as a mammalian cell (including but not limited to HeLa cells and 293T cells), insect cell (including but not limited to sf9 cells). In this aspect of the invention, preferably, modulating the level of acetylation of ORF2 is enhancing the level of acetylation of ORF 2. In one embodiment, modulating the level of acetylation of ORF2 is achieved by inhibiting HDAC6 expression and/or activity in a cell. In further embodiments, HDAC6RNAi molecules (e.g., siRNA) are used to knock down the level of HDAC6 expression in cells. In a further embodiment, the HDAC6 activity is decreased in a cell using a chemical inhibitor of HDAC 6. In another embodiment, the stability of ORF2 in a cell is increased, and/or the amount of ORF2, in particular ORF2 multimers, in a cell is increased, by modulating the level of acetylation at position K411 of ORF 2. In another embodiment, replication of the virus in the cell is increased by modulating the level of acetylation at position K411 of ORF 2. In another embodiment, the assembly of VLPs and/or the production of VLPs in a cell is increased by modulating the level of acetylation at position K411 of ORF 2.

In another aspect, the invention also provides an isolated K411 acetylated HEV ORF2 polypeptide. The ORF2 polypeptide comprises a HEV genotype 1-4 wild-type ORF2 sequence, or homolog or variant thereof. In a further aspect, the invention provides methods and cell cultures for increasing production of K411 acetylated HEV ORF2 polypeptide, characterized by inhibiting expression and/or activity of HDAC6in a cell, wherein production of K411 acetylated ORF2 polypeptide in the cell is increased relative to a control cell that does not inhibit HDAC6 expression and/or activity. In one embodiment, the cell is an HDAC6 knockdown cell. In another embodiment, the HDAC6 chemical inhibitor is administered to a cell. In one embodiment, the method of the present invention further comprises: collecting the produced acetylated HEV ORF2 polypeptide or the mixture of the K411 acetylated HEV ORF2 polypeptide and the non-acetylated HEV ORF2, and using the mixture to prepare immune compositions or vaccines or HEV virus-like particles. In one embodiment, the invention also provides the use of the acetylated HEV ORF2 polypeptide to promote multimerization of ORF2 and/or formation of a virus or virus-like particle.

In yet another aspect, the invention provides a method of treating HEV infection comprising administering to an individual infected with HEV an HEV ORF2 polypeptide K411 acetylation modulator. In one embodiment, the modulator reduces the level of acetylation of ORF2 polypeptide K411. In further embodiments, the modulator increases the expression and/or activity of HDAC6in a cell. In further embodiments, the modulator is an HDAC6 expression nucleic acid.

Brief Description of Drawings

FIG. 1 acetylation of HEV capsid protein ORF2 at conserved residue K411.

(A) 293T cells expressing GFP-1-ORF2(112-660) or GFP-4-ORF2(112-660) were immunoprecipitated with a GFP antibody and stained by Coomassie blue.

(B-D) Mass Spectrometry to identify acetylation at the K411 position. The GFP-1-ORF2(112-660) (panel C) and GFP-4-ORF2(112-660) proteins expressed in 293T cells were analyzed by mass spectrometry (panel D). EPTVK⁴¹¹The LYTSVEN peptide is conserved between different HEV genotypes (FIG. 1B).

(E) Acetylation of GFP-1-ORF2 and GFP-4-ORF2 was determined by immunoblotting in 293T cells. GFP-1-ORF2(112-660), GFP-4-ORF2(112-660) or GFP, which was overexpressed in 293T cells, was immunoprecipitated by an anti-GFP antibody, followed by western blot detection using an anti-acetyl-lysine (acetyl-Lys) antibody (left panel) and an anti-GFP antibody (right panel).

(F) Immunoblotting revealed that K411 is the main acetylation site for GFP-1-ORF2(112-660) and GFP-4-ORF2 (112-660). Overexpression of GFP-1-ORF2 in 293T cells^K411R(112-660),GFP-4-ORF2^K411R(112-660) and wild-type plasmid. Following immunoprecipitation using anti-GFP antibody, immunoprecipitates were determined by anti-acetyl lysine antibody (top panel) and anti-GFP antibody (bottom panel). WT: a wild type; K411R: K411R mutant.

FIG. 2: the K411R mutation of ORF2 hinders HEV inclusion body formation.

(A) Representative immunofluorescence plots of GFP-1-ORF2(112-660) wild type (upper panel) and K411R mutant (lower panel) in HeLa cells. GFP (green) and immunofluorescence stain α -tubulin (red). DNA was stained with DAPI (blue). Note that GFP-1-ORF2 is localized in the viral inclusion bodies and cytoplasm. (scale bar: 10 μm).

(B) The same experiments as described in A were performed for GFP-4-ORF2 (112-660).

(C) For GFP-1-ORF2(1-660), GFP-1-ORF2(112-660), GFP-1-ORF2^K411R(112- > 660) the same experiment as described in A was performed.

(D) HeLa cells transiently expressing HA-tagged or Flag-tagged 1-ORF2(112-660) were fixed and immunostained with anti-HA or Flag antibody (green), anti-a-tubulin antibody (red), and DAPI (blue). (scale bar: 10 μm).

(E) Cells containing viral inclusion bodies were analyzed statistically in cells transfected with GFP-1-ORF2(112-660) and GFP-4-ORF2 (112-660). Three independent experiments were performed, with n being 200 cells/group. Spot cells: number of cells having inclusion body in cytoplasm (GFP dot number > 3/cell)

(F) The full length molecules of GFP-1-ORF2(1-660), GFP-1-ORF2(112-660) and GFP-1-ORF2^K411R(112-660) cells containing inclusion bodies were analyzed statistically in transfected cells. Three independent experiments were performed, n-200 cells/group; p<0.01. Data are mean ± SD, using Student's t-test. Spot cells: number of cells having inclusion body in cytoplasm (GFP dot count)>3/cell).

FIG. 3: the effect of K411R on inclusion body formation was assessed dynamically.

(A-B) on GFP-1-ORF2(112-660) and GFP-1-ORF2 in HeLa cells^K411R(112-660) imaging of live cells.

The GFP-1-ORF2 plasmid was transfected with the RFP-tagged histone 2B plasmid. 12 hours after transfection, 5 images along the z-axis of living cells were obtained separately and the final image was obtained by merging the image layers. It was noted that viral inclusion bodies gradually appeared in the wild-type group and began early. While GFP-1-ORF2^K411R(112-660) during this time course, viral inclusion bodies cannot be efficiently formed.

FIG. 4: HDAC6 deacetylates ORF2 and affects IB formation.

(A) Flag-HDAC6 interacts with GFP-1-ORF2 and GFP-4-ORF 2. 293T cells were transfected with the GFP-ORF2(112-660) plasmid and the Flag plasmid or the Flag-HDAC6 plasmid and immunoprecipitated with Flag antibody, followed by western blotting with GFP antibody and Flag antibody. Results of western blotting of cell lysates are also shown. In the figure, "Flag" indicates the transfection Flag plasmid; "Flag-HDAC" means the transfection Flag-HDAC6 plasmid; "1-ORF 2" means the transfection of the GFP-1-ORF2(112-660) plasmid; "4-ORF 2" indicates the transfection of the GFP-4-ORF2(112-660) plasmid.

(B) GFP-1-ORF2 and GFP-4-ORF2 interact with endogenous HDAC 6. 293T cells were transfected with GFP, GFP-1-ORF2(112-660) or GFP-4-ORF2(112-660) plasmids, immunoprecipitated with rabbit GFP antibody, and western blots with HDAC 6-specific antibodies and mouse GFP antibody. Results of western blotting of cell lysates are also shown.

(C) HDAC6 knockdown increased acetylation of GFP-1-ORF2 and GFP-4-ORF 2. The GFP-1-ORF2(112-660) and GFP-4-ORF2(112-660) plasmids were expressed in HDAC6 knock-down 293T cells. Following immunoprecipitation using GFP antibody, acetylated ORF2 was detected by western blot using anti-acetyl lysine antibody. Western blot detection using anti-HDAC 6, anti-GAPDH and anti-GFP antibodies on cell lysates and immunoprecipitates is also shown. "con" in the figure indicates transfection of nonspecific siRNA as a control "; "HDAC 6 i" means the transfection of HDAC6 siRNA.

(D) Immunoblots showed that acetylation of GFP-1-ORF2 and GFP-4-ORF2 increased with TBSA treatment. GFP-1-ORF2(112-660) and GFP-4-ORF2(112-660) plasmids were overexpressed in 293T cells and treated with DMSO or TBSA. Following immunoprecipitation using GFP antibody, the immunoprecipitates were detected by anti-acetyl lysine antibody (top panel) and anti-GFP antibody (bottom panel).

(E) Representative immunofluorescence profiles of GFP-1-ORF2(112-660) under HDAC6inhibitor treatment. Trichostatin A (TSA, 0.5. mu.M) or Tubastatin A (TBSA, 3. mu.M) was added and treated for 8 hours.

(F) Representative immunofluorescence profiles of GFP-4-ORF2(112-660) under HDAC6inhibitor treatment. Trichostatin A (TSA, 0.5. mu.M) or Tubastatin A (TBSA, 3. mu.M) was added and treated for 8 hours.

(G) Cells containing more than 10 viral inclusion bodies were statistically analyzed in GFP-1-ORF2(112-660) transfected cells treated with DMSO, TSA or TBSA. Three independent experiments were performed, with n being 200 cells/group. P <0.01, p < 0.001. Data are mean ± SD, using Student's t-test.

(H) Cells containing more than 10 viral inclusion bodies were statistically analyzed in GFP-4-ORF2(112-660) transfected cells treated with DMSO, TSA or TBSA. Three independent experiments were performed, with n being 200 cells/group. P <0.01, p < 0.001. Data are mean ± SD, using Student's t-test.

FIG. 5: inhibition of HDAC6 promotes stability of HEV ORF 2.

(A) HDAC6RNAi upregulated HEV ORF2 polypeptide levels. 293T cells were transfected with HDAC6siRNA or control siRNA (Santa cruz) for 48 h and with GFP-1-ORF2 or GFP-4-ORF2(112-660) for 24 h. Cell lysates were collected and detected by western blot using anti-HDAC 6 antibody, anti-GFP antibody and anti-GAPDH antibody.

(B) The nonacetylated mutant of HEV ORF2 reduced the protein stability of ORF2 via a proteasome-dependent pathway. Using GFP-1-ORF2(112-660) WT (wild type), GFP-1-ORF2^K411R(112-660), GFP-4-ORF2(112-660) WT (wild type), and GFP-4-ORF2^K411R(112-660) 293T cells were transfected for 24 hours. Cell lysates were collected and detected by western blotting with anti-GFP antibody and anti-GAPDH antibody. MG132 was added and the treatment was carried out for 4 hours. It was noted that the rapid degradation of the non-acetylating mutant of ORF2 could be rescued by the addition of the proteasome inhibitor MG 132. The results obtained for different western blot development exposure times are shown in the figure.

(C) Tubastatin A increases the stability of GFP-ORF2 polypeptide. 293T cells were transfected with GFP-1-ORF2(112-660) and GFP-4-ORF2(112-660) for 24 h. Tubastatin A (TBSA, 3. mu.M) was added and treated for 8 hours. The sample concentration used in non-reducing PAGE was twice that used in reducing SDS-PAGE, but from the same source.

FIG. 6: acetylation of ORF2K411 can modulate the stability of ORF2 and HEV IB formation.

(A) Tubastatin A increased HEV antigen levels in the supernatant of PLC/PRF/5 cell cultures. After 3 weeks of HEV infection, DMSO, Trichostatin A (TSA, 0.5. mu.M) or Tubastatin A (TBSA, 3. mu.M) was added to the PLC/PRF/5 cells, and after 3 days, cell culture supernatants were collected and tested for HEV antigen by ELISA. P < 0.001. Data are mean ± SD, using Student's t-test. In the figure, "HEV Ag S/Co" is a unit used in ELISA detection, and represents: sample OD/cut off detection line value.

(B) ORF2 VLPs expressed in sf9 cells were detected by coomassie blue staining in non-denaturing PAGE and analyzed by mass spectrometry.

(C-E) TSA, TBSA and CAY improved the expression of ORF2 in sf9 cells. DMSO, Trichostatin A (TSA, 1. mu.M), Tubastatin A (TBSA, 6. mu.M) or CAY10603(CAY, 0.2. mu.M) was added and the mixture was treated for 5 days. The sf9 cell culture supernatant was collected and ultracentrifuged at 40000rpm for 2 hours and whole cell culture supernatant and free fraction and VLP fraction were examined by ELISA. P <0.05, p <0.01, p < 0.001. Data are mean ± SD, using Student's t-test.

(F) Transmission electron microscopy detection of Wild Type (WT)0GFP-1-ORF2, GFP-1-ORF2, and mutant GFP-1-ORF2 in cells treated with TBSA or DMSO^K411R. The red circle indicates the VLP region.

(G) Transmission electron microscopy detection of Wild Type (WT) GFP-4-ORF2, GFP-4-ORF2, and mutant GFP-4-ORF2 in cells treated with TBSA or DMSO^K411R. The red circle indicates the VLP region.

FIG. 7: 1-ORF and 4-ORF and truncated fragments thereof and HDAC6 sequence.

FIG. 8: representative immunofluorescence images of HEV ORF2 in a549 cells. Representative immunofluorescence images of GFP-1-ORF2(112-660) (green) wild-type and K411R mutant in A549 cells with or without TSA treatment. DNA was stained with DAPI (blue). It was noted that upon TSA treatment, the number of inclusion bodies formed in the cells increased for wild-type ORF2, due to the inhibition of deacetylation of ORF 2. In contrast, under TSA treatment, the ORF2 mutant was unaffected, yet dispersed predominantly uniformly in the cytoplasm.

FIG. 9: the proportion of viable spot cells transfected with GFP-ORF2 was statistically analyzed. Calculation of GFP-1-ORF2(112-660), GFP-1-ORF2^K411R(112-660), GFP-4-ORF2(112-660) and GFP-4-ORF2^K411R(112-660) transfected spot HeLa cells. Three independent experiments were performed, n 200 cells/group; p<0.01,***p<0.001. Data are mean ± SD, using Student's t-test. Spot cells: cells having inclusion bodies in cytoplasm (GFP dot count)>3/cell)。

FIG. 10: live cell imaging of GFP-4-ORF2(112-660) and GFP-4-ORF2(112-660) K411R in HeLa cells. (A-B) GFP-4-ORF2(112-660) and GFP-4-ORF2 in HeLa cells^K411R(112-660) imaging of live cells. The GFP-4-ORF2 plasmid and the RFP-tagged histone 2B plasmid were transfected together. 12 hours after transfection, 5 images were taken along the living cell z-axis and the layers were combined to obtain the final image. It was noted that viral inclusion bodies gradually appeared in the wild-type group, starting early. GFP-4-ORF2^K411R(112-660) failed to efficiently form viral inclusion bodies over the course of time.

FIG. 11: 293T cells treated with DMSO, TBSA or TSA 24 h after transfection with GFP-1-ORF 2. 293T cells were transfected with GFP-1-ORF2 plasmid, added with DMSO, Trichostatin A (TSA, 0.5. mu.M) or Tubastatin A (TBSA, 3. mu.M), treated for 8 hours, and observed under a microscope. The results show that the cell state of the Trichostatin A or Tubastatin A treated group is not significantly different from that of the DMSO control group.

FIG. 12: the GFP-1-ORF2(112-660) and GFP-4-ORF2(112-660) protein levels were upregulated by TSA treatment.

FIG. 13: degradation of GFP-1-ORF2 is dependent on the proteasomal pathway. 293T cells transfected with GFP-1-ORF2 were treated with cycloheximide (CHX, 10. mu.M) and MG132 (50. mu.M) for 0-9 hours. Immunoblotting examined the amount of GFP-1-OFR2 and actin. The ORF2 polypeptide in the CHX and DMSO-treated groups decreased in protein amount as the duration of drug action was prolonged. ORF2 polypeptide in CHX and MG132 treated groups showed no significant change in protein amount with increasing duration of drug action.

Detailed Description

The inventors have found that ORF2 is acetylated at conserved amino acid residue 411 in Hepatitis E Virus (HEV) of genotypes 1 and 4 (example 1). Prolonged live cell imaging of wild-type and acetylated mutant ORF2 indicated that K411 acetylation plays a critical role in hepatitis e Inclusion Body (IB) self-assembly (examples 2 and 3). Furthermore, the inventors found that K411 acetylation can enhance the resistance of HEV ORF2 polypeptide to proteasome-dependent degradation, increasing the stability of HEV ORF2 polypeptide (example 5). Further, the inventors demonstrated that HDAC6 (histone deacetylase 6) catalyzes the deacetylation of K411-acetylated HEV ORF2 (example 4), negatively modulates viral Inclusion Body (IB) formation (example 4) and ORF2 polypeptide stability (example 5). Also, the inventors demonstrated that inhibition of HDAC6 can increase HEV viral replication and virus-like particle formation (example 6). These findings by the present inventors provide direct evidence that HEV virus utilizes a novel mechanism of post-translational host modification to maintain the stability of capsid protein ORF2 and enhance viral inclusion formation, suggesting that HDAC6 exerts a host defense mechanism against HEV infection.

Thus, based on the above findings, the present inventors proposed:

(a) an isolated K411 acetylated ORF2 polypeptide and its use for promoting assembly of a virus or virus-like particle.

(b) A method for improving the production of hepatitis E virus, virus-like particles (VLPs) and ORF2 polypeptides by modulating the acetylation of ORF2 at position K411; and methods of improving the stability of HEV OFR2 protein;

(c) use of a modulator of K411 acetylation level of ORF2, in particular an HDAC6inhibitor, in increasing HEV viral replication and virus-like particle formation and ORF2 stability in a cell;

(d) a novel method of treatment of HEV which comprises administering to a HEV patient, such as a critically ill patient and a chronic HEV-infected patient, or a non-human animal infected with HEV (such as a pig, to reduce the amount of HEV virus excreted into the environment by said animal), an agent and/or means for modulating K411 acetylation of HEV ORF2 (e.g., reducing or inhibiting ORF2 acetylation, or enhancing deacetylation of ORF2 by HDAC 6).

In any of the above aspects, preferably the HEV virus is a HEV virus of genotype 1-4, more preferably genotype 1or 4. Preferably, the ORF2 polypeptide comprises the peptide sequence E (P/L) TVK⁴¹¹LYTSVEN, and acetylated at the conserved residue K411 of the peptide. Preferably, the level of acetylation of ORF2 at K411 is increased by inhibiting HDAC6in the cells used for production. Without being bound by theory, K411 acetylation of ORF2 may facilitate ORF2 multimerization, and/or increase the resistance of HEV ORF2 polypeptides to the proteasome degradation pathway and the stability of ORF 2. Without being bound by any theory, by increasing K411 acetylation of ORF2To increase the amount of ORF2 polypeptide in the cell for virus assembly or VLP assembly and correspondingly increase the yield of virus or VLP.

Aspects of the invention are described in further detail in the following subsections.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory procedures, as used herein, are conventional procedures that are widely used in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

Defining:

in the present invention, the term "hepatitis E virus" ("HEV") refers to a virus, virus type, or virus class. HEV is assigned to a single-stranded positive-stranded RNA icosahedral virus of the hepacivirus genus, has a genome size of 7.2kb, and comprises three Open Reading Frames (ORFs), wherein ORF2 encodes a capsid protein. There are four major genotypes of HEV, namely 1, 2,3 and 4. In the present invention, the ORF2 polypeptide of HEV virus preferably comprises the peptide sequence EPTVK⁴¹¹The LYTSVEN, preferably HEV virus, is a virus of genotype 1-4, more preferably a virus of genotype 1or 4.

In the present invention, the terms "HEV ORF 2" or "ORF 2 polypeptide" or "ORF 2" are used interchangeably to refer to the capsid protein encoded by the ORF-2 open reading frame from the Hepatitis E Virus (HEV) genome, or a fragment thereof, or a homolog or variant thereof. The ORF2 polypeptide of the invention is characterized by comprising the consensus sequence: e (P/L) TVK⁴¹¹LYTSVEN, preferably comprising the consensus sequence: EPTVK⁴¹¹LYTSVEN, capable of being acetylated at K411.

The capsid ORF2 protein of HEV virus and its sequence are well known in the art. ORF2 of the invention can comprise an ORF2 sequence from genotype 1-4 HEV, such as the ORF2 sequence of genotype 1 (also referred to simply as 1-ORF2) or the ORF2 sequence of genotype 4 (also referred to simply as 4-ORF 2). An exemplary ORF2 of the invention is HEV ORF2 protein of genotype 1 given under GenBank accession number JQ655734 (sequence shown in SEQ ID NO: 1). Another exemplary ORF2 of the invention is genotype 4 HEV ORF2 protein (sequence shown in SEQ ID NO:2) given under GenBank accession number JQ 655736. Other HEV ORF2 suitable for use in the present invention include, for example, the sequences given under the following accession numbers: p29326; p33426; Q6J8F 7; q9IVZ 8; q9YLQ 9; q03500; q04611; q68985; q81871.

In the present invention, the expression "ORF 2 fragment" or "truncated fragment of ORF 2" refers to a fragment of ORF2 that is truncated at the N-terminus and/or C-terminus relative to the full-length protein of ORF 2. In an exemplary embodiment, the HEV ORF2 fragment of the invention consists of amino acid residues 112-660 of the ORF2 protein under SEQ ID NO: 1or 3 or any of the aforementioned accession numbers, or consists of amino acid residues 112-607 or 112-608. The ORF2 fragment retained the consensus sequence: e (P/L) TVK⁴¹¹LYTSVEN, and the nature of acetylation at K411, and preferably retains the ability to multimerize and assemble into viral capsids or virus-like particles. Preferably, the fragment is immunogenic.

According to the present invention, the term "variant" when used in the context of a protein/polypeptide refers to a protein whose amino acid sequence has one or more (e.g., 1-10 or 1-5 or 1-3) amino acid differences (e.g., conservative amino acid substitutions) from the amino acid sequence of a reference protein/polypeptide (e.g., HEV capsid protein ORF2 of the present invention), or at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, and which retains the essential properties of the reference protein/polypeptide. In the present invention, the essential property of a protein/polypeptide (e.g., for variants of the HEV capsid proteins of the present invention) may refer to the ability to acetylate at K411, and preferably to multimerize and assemble into viral capsids or virus-like particles.

In the present invention, when referring to amino acid positions, it is determined by reference to the amino acid sequence of HEV ORF2 protein (also referred to as 1-ORF2) of SEQ ID NO:1, i.e., accession number JQ 655734. Can be obtained by performing an amino acid sequence alignment (e.g., using BLAST; fromhttp://blast.ncbi.nlm.nih.gov/Blast.cgiPROGRAM＝blastp&PAGE_ TYPE＝BlastSearch&LINK_LOC＝blasthomeObtained Basic Local align nment Search Tool, aligned using default parameters), the corresponding amino acid positions on other ORF2 polypeptides (including the full-length sequence or ORF2 fragment) were identified. Thus, in the present invention, reference to "K411" refers to amino acid residue 411 of SEQ ID NO. 1, or amino acid residues aligned at corresponding positions on other HEV ORF2 sequences. Accordingly, in the present invention, when referring to a "corresponding sequence fragment" or a "corresponding fragment" is intended a fragment at an equivalent position (i.e., the corresponding amino acid position) in the sequences being compared when the sequences are optimally aligned, i.e., when the sequences are aligned for the highest percent identity.

In the present invention, the expression "acetylation" of K411 is used interchangeably with "acetylation" in reference to the ORF2 polypeptide, and indicates that the polypeptide has an acetylated lysine residue at the K411 position (i.e. the position corresponding to the K411 position of the reference SEQ ID NO:1 sequence). Accordingly, the expression "non-K411 acetylated" is used interchangeably with "non-acetylated" in reference to an ORF2 polypeptide, indicating that the polypeptide does not have acetylation at the K411 position. Acetylation of K411 on the ORF2 polypeptide can be determined by a variety of means known to those skilled in the art, such as mass spectrometry, western blotting, and the like.

In the present invention, HDAC6 refers to histone deacetylase 6 of the histone deacetylase IIb family. HDAC6 has two catalytic domains and a zinc finger domain. HDAC6 can catalyze deacetylation of K411 acetylated HEV ORF2 polypeptides (e.g., SEQ ID NOS: 1-4) to produce K411 deacetylated ORF2 polypeptides. The sequence of HDAC6 is known in the art and is available from GenBank. An exemplary HDAC6 protein sequence and its coding sequence are listed in FIG. 7 (SEQ ID NOS: 5 and 6).

According to the invention, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When both sequences being compared are occupied at a position by the same base or amino acid monomer subunit (e.g., both ORF2 polypeptides are occupied by lysine at position K411), then the molecules are identical at that position. In the context of comparing two nucleic acid or polypeptide sequences, a percent "identity" refers to two sequences having a specified percentage of identical nucleotides or amino acid residues (e.g., at least 60% identity, optionally at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity within a specified region) when compared and aligned for maximum correspondence using one of the following sequence comparison algorithms or by manual alignment and visual inspection over a comparison window or within a specified region. Alternatively, the percent identity may be any integer from 60% to 100%.

"percent sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window in which a portion of a polynucleotide sequence or polypeptide sequence can contain additions or deletions (i.e., gaps) as compared to a portion of a reference sequence (which does not contain additions or deletions) after optimal alignment of the two sequences. The percentage is calculated by: determining the number of positions of identical nucleic acid bases or amino acid residues present in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

Methods of sequence alignment for comparison are well known in the art. Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms (see Altschul et al, Nuc. acids Res.25: 3389-.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the biological function of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Typical conservative amino acid substitutions are those in which one amino acid is substituted for another with similar chemical properties (e.g., charge or hydrophobicity). The following six groups each contain amino acids that can be typically conservatively substituted for each other: 1) alanine (a), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

The term "isolated" or "substantially purified" means a chemical composition that is substantially free of other cellular components. Purity and homogeneity can generally be determined by techniques of analytical chemistry such as polyacrylamide gel electrophoresis or high performance liquid chromatography or mass spectrometry. Generally, a substantially purified or isolated protein should comprise more than 70% of all macromolecular species present in the preparation. In some embodiments, the purified protein comprises greater than 90%, greater than 95%, or is purified to substantial homogeneity of all macromolecular species present, wherein no other macromolecular species are detected by conventional techniques. For the isolated acetylated ORF2 proteins of the invention, in some embodiments, the protein is at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% or more pure, and preferably no unacetylated ORF2 protein at K411 is detectable by conventional techniques.

According to the present invention, the term "virus-like particle (VLP)" refers to an empty shell structure, free of viral nucleic acids, structurally similar to a native viral particle, which is typically composed of viral capsid proteins or variants or fragments thereof. VLPs are highly immunogenic and immunoreactive because they are structurally very similar to native viral particles. At the same time, VLPs are not infectious because they do not contain the genetic material of the virus. Due to the above advantages, VLPs have been developed for use as vaccines. Furthermore, VLPs structurally allow insertion of foreign genes or gene fragments to form chimeric VLPs and are capable of displaying foreign antigens on their surface. Furthermore, most VLPs also have the ability to encapsulate nucleic acids or other small molecules, and thus, they can also serve as carriers of genes or drugs.

According to the present invention, the term "assembly" refers to the process of forming a regular granular structure between structural proteins (e.g. capsid proteins) of a virus or between a structural protein and a nucleic acid through various interactions, which includes the assembly of native viral particles and the assembly of virus-like particles. ORF2 can be automatically assembled in insect and mammalian cells to produce VLP particles. For ORF2 produced by the e.coli system, VLP particles can be produced by assembly from ORF2 in a VLP assembly solution. Assembly solutions for assembly from ORF2 to form HEV virus-like particles are known in the art.

In the present invention, an "expression construct" refers to a recombinant nucleic acid molecule capable of expressing a polypeptide of interest. For example, a "recombinant ORF2 expression construct" is a recombinant nucleic acid molecule capable of expressing an ORF2 polypeptide. It is understood that expression constructs encompass vector constructs.

Acetylated HEV ORF2 protein and application thereof

The native HEV ORF2 protein is a protein comprising 660 amino acids. Native ORF2 is synthesized as a precursor in cells infected with HEV and processed by signal sequence cleavage into a mature protein capable of self-assembly. When the ORF2 coding sequence was expressed in Sf9 insect cells using the baculovirus system, stable protein products with estimated molecular weights of 72kDa and 59-62kDa could be produced. Truncated proteins comprising amino acids between positions 125-601 and 112-607 and 112-660 can be assembled into virus-like particles (Surjit, M. et al, Journ. Virol, 78: 320-328(2004), Li, TC et al, Journ. Virol.79,12999-13006 (2005)). A neutralizing domain is present between amino acid residues aa458-aa607 of HEV ORF2 (Zhou, YH et al, Vaccine 22: 2578-2585(2004), Ahmad, I. et al, Virus Res.161: 47-58 (201)).

The present invention is based, in part, on the observation that HEV ORF2 protein is acetylated at the K411 position, and that acetylation at this position can promote stability of ORF2 and dimerization and multimerization of ORF2, and promote assembly of viral particles and VLP particles.

Accordingly, in one aspect, the invention provides an isolated HEV ORF2 polypeptide characterized by an acetylation at the K411 position. In thatIn one embodiment, the ORF2 polypeptide of the invention comprises the peptide sequence E (P/L) TVK⁴¹¹LYTSVEN, preferably comprising the peptide sequence: EPTVK⁴¹¹LYTSVEN. In a further embodiment, the ORF2 polypeptide of the invention is an ORF2 fragment comprising the amino acid sequence starting between position 112-125 and ending between position 601-660 or 607-660. In yet another embodiment, the HEV ORF2 polypeptide of the invention is an ORF2 fragment comprising the C-terminal amino acid residues at positions 458-607 and the N-terminal amino acid residue at position 125-457 to enable VLP formation and is immunogenic. In a preferred embodiment, the HEV ORF2 fragment of the invention comprises at least the amino acid residues at position 125-601, preferably 125-607. In yet another preferred embodiment, the HEV ORF2 fragment of the invention lacks the N-terminal amino acid sequence of aa 1-111. FIG. 1 shows exemplarily the full-length sequence (SEQ ID NO:1) and the aa112-660 truncated fragment (SEQ ID NO:2) of a HEV ORF2 genotype 1 protein, and the full-length sequence (SEQ ID NO:3) and the aa112-660 truncated fragment (SEQ ID NO:4) of a HEV ORF2 genotype 4.

It is understood by those skilled in the art that mutations or variations (including, but not limited to, substitutions, deletions and/or additions) can be naturally occurring or artificially introduced into the HEV ORF2 polypeptides (including full-length sequences and truncated fragments) of the invention without affecting their biological function. Thus, in a further aspect, the invention provides an isolated HEV ORF2 polypeptide, wherein the HEV ORF2 polypeptide comprises a native (i.e., wild-type) HEV ORF2 polypeptide (full-length sequence or fragment as previously described), or a sequence homologous thereto, and preferably biologically equivalent to a native HEV ORF2 polypeptide. In the present invention, "biologically equivalent" means that the polypeptide is capable of being acetylated at the K411 position and capable of being assembled to form a viral capsid or virus-like particle, and is preferably immunogenic. Thus, in some embodiments, when a vaccine composition of the invention comprising an HEV ORF2 polypeptide of the invention and optionally a pharmaceutically acceptable carrier is injected into a mammal, it stimulates the production of protective antibodies that protect the mammal from wild-type HEV. In the present invention, preferably, the identity of the homologous sequence to the native HEV ORF2 polypeptide sequence is preferably 70% or more, more preferably 80% or 90% or more, particularly preferably 95% or more, for example, 95%, 96%, 97%, 98%, 99% or more. More preferably, the homologous sequence differs from the native HEV ORF2 polypeptide sequence by only 1-10 amino acid changes, and preferably 1-5, or 1-3 amino acid changes, more preferably conservative amino acid substitutions.

It is known to those skilled in the art that various fragments of ORF2 can be used for in vitro and in vivo assembly to form VLP virus-like particles (see WO2010099547, and Expression and self-assembly of Expression virus-like particles of hepatites E virus. Li TC, Yamakawa Y, Suzuki K, Tatsumi M, Razak MA, Uchida T, Takeda N, Miyamura T., J Virol.1997 Oct; 71(10):7207-13. Expression elements of the cap protein for self-assembly inter-Expression virus-like particles of hepatites E virus. TC, Takeda N, Miyamura T, Matura Y, JC, aging H, Hamming L20012999. J., J12979: 79). These ORF2 fragments are also suitable for use in the present invention. Thus, in some embodiments, the invention provides ORF2 fragments that are acetylated at position K411. In some embodiments, the ORF2 fragment has a deletion of at least 25 consecutive amino acids from the N-terminus, preferably at least 50 amino acids, at least 100 amino acids, preferably the amino acid sequence from position 1 to 111 from the N-terminus. In some embodiments, the ORF2 fragment has a deletion in the C-terminal region, e.g.deletion of at least 10 consecutive C-terminal amino acid residues, e.g.at least 20, preferably C-terminal amino acid residues at positions 602-, 660,603-, 604-, 605-, 606-, 660,607-, 660,608-660, or 609-660.

In some preferred embodiments, the present invention provides an isolated acetylated hepatitis E virus open reading frame 2(HEV ORF2) polypeptide characterized by comprising the peptide sequence E (P/L) TVK⁴¹¹LYTSVEN, and has acetylation at K411. Preferably, the polypeptide comprises a sequence selected from: (a) the full-length sequence of wild-type HEV virus ORF2 protein, preferably genotypes 1-4, more preferably genotypes 1 and 4, of HEV ORF2 protein; (b) a truncated fragment of (a), said fragment comprising E (P/L) TVK⁴¹¹The LYTSVEN peptide sequence, preferably retains the ability to multimerize and assemble into viral capsids or virus-like particles, preferably,the fragments are immunogenic; (c) a homologue or variant of (a) or (b). In some embodiments, the ORF2 fragment is truncated with the amino acid sequence of N-terminal a1-aa111, preferably comprising at least the aa125 to 601, or 125 to aa607 region of the full-length ORF2 sequence, preferably consisting of the amino acid sequence of aa112 to aa 660.

In a preferred embodiment, the ORF2 polypeptide comprises the full length sequence of native HEV ORF2 from genotype 1or a truncated fragment thereof, or a sequence homologous thereto. In yet another preferred embodiment, the ORF2 protein comprises the full length sequence of native ORF2 from genotype 4 or a truncated fragment thereof, or a sequence homologous thereto. In still other preferred embodiments, the ORF2 polypeptide comprises a sequence selected from SEQ ID NOS 1-4, or a sequence homologous thereto. Preferably, the homologous sequence has at least 90%, 95%, 96%, 97%, 98%, 99% or more identity to the sequence of SEQ ID NO. 1or 2, more preferably differs from the sequence of SEQ ID NO. 1or 2 by only 1-10 amino acid changes, and preferably 1-5, or 1-3 amino acid changes, more preferably the changes are conservative amino acid substitutions. Preferably, the homologous sequence has at least 90%, 95%, 96%, 97%, 98%, 99% or more identity to the sequence of SEQ ID NO. 3 or 4, more preferably differs from the sequence of SEQ ID NO. 3 or 4 by only 1-10 amino acid changes, and preferably 1-5, or 1-3 amino acid changes, more preferably the changes are conservative amino acid substitutions. In these embodiments, preferably, the truncated fragment is an N-terminal truncated fragment, e.g., lacking the amino acid sequence aa1-aa111 at the N-terminus. More preferably, the fragment comprises at least the region from amino acid 125 to amino acid 601, preferably from amino acid 112 to 660 of the full-length ORF2 sequence.

The HEV ORF2 acetylated polypeptides of the present invention can be readily obtained by expressing DNA fragments encoding these proteins and fragments thereof in cells with an acetylation system, such as mammalian cells, insect cells, and e. Suitable expression systems, such as baculovirus-based, mammalian cell-based expression systems, are known in the art. Such expression systems are widely described in the art and they are commercially available. Baculovirus-based is a preferred system in view of ease of use and relatively high yield. In some embodiments, the expressed protein lacks the N-terminal 111 amino acids of ORF2 and is soluble. In still other embodiments, a recombinant ORF2 polypeptide, such as ORF2 fragment aa112-aa607 or aa112-660, is secreted in the culture medium as a soluble protein but is also present in the cell pellet.

Methods for separating acetylated proteins from non-acetylated proteins are known in the present invention (see e.g. CN103930433A and US2004247614) and include e.g. chromatography, HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography or other purification techniques. For example, to purify and isolate the acetylated proteins of the invention, the ORF2 protein may be enriched first with the ORF2 antibody and then the acetylated lysine antibody enriched with the acetylated modified ORF2 protein. In addition, protein purification can be facilitated by N-terminal or C-terminal tag (e.g., Flag) design. Furthermore, an antibody specific to K411 acetylated ORF2 protein may be prepared and purified using the specific antibody. In one embodiment, the invention also provides a composition comprising a purified K411 acetylated ORF2 protein.

The inventors found that when an acetylated ORF2 polypeptide of the invention is produced in a cell, the level of acetylation of the polypeptide of the invention at position K411 is affected by the HDAC6 deacetylase activity of the cell. Thus, in one aspect, the invention also provides a method of increasing production of an acetylated HEV ORF2 polypeptide, comprising: introducing a nucleic acid encoding a HEV ORF2 polypeptide into a host cell, wherein the cell is an HDAC6 knockdown cell; culturing the cell under conditions suitable for expression of the nucleic acid. Preferably, the HDAC6 knockdown cell is a HDAC6 conditional knockdown cell. In yet another aspect, the invention provides a method of increasing production of an acetylated HEV ORF2 polypeptide, comprising: introducing a nucleic acid encoding HEV ORF2 protein or fragment thereof into a host cell; culturing the cell under conditions suitable for expression of the nucleic acid; administration of an HDAC6inhibitor to a cell inhibits HDAC6 activity. Preferably, the HDAC6inhibitor is an HDAC6 chemical inhibitor, such as a non-selective inhibitor like TSA or a selective inhibitor like TBSA, more preferably an HDAC6 selective inhibitor. In one embodiment, the cell is a HEV virus-infected cell. In one embodiment, the cell is a recombinant cell into which an ORF2 expression construct has been introduced. In one embodiment, the production of K411 acetylated ORF2 protein in a cell is increased relative to a control cell that does not inhibit HDAC6 expression and/or activity. In one embodiment, the method of the present invention further comprises: collecting the produced acetylated HEV ORF2 protein, or a mixture of the K411 acetylated HEV ORF2 protein and the non-acetylated ORF 2. In one embodiment, the HEV ORF2 polypeptide is assembled in a cell to produce VLPs. In some embodiments, an isolated K411 acetylated ORF2 polypeptide or a K411 acetylated ORF2 polypeptide obtained according to the methods of the invention or a mixture thereof with non-acetylated ORF2 is used for the preparation of an immunological composition or vaccine, or for the preparation of VLPs of virus-like particles.

In another aspect, the invention provides a cell culture for producing a K411 acetylated ORF2 polypeptide of the invention, comprising HDAC6 knockdown cells comprising a nucleic acid expressing an ORF2 polypeptide. In one embodiment, the cell is a HEV virus-infected cell. In one embodiment, the cell is a cell into which a recombinant ORF2 expression construct has been introduced. In some embodiments, the cell is used to produce an acetylated ORF2 polypeptide, virus, or virus-like particle.

In addition, the invention also provides dimers or multimers of the K411 acetylated ORF2 protein of the invention. Without being bound by theory, it is believed that acetylation of K411 has the ability to promote aggregation and assembly of the ORF2 protein. Thus, in a further aspect, the invention also provides the use of the K411 acetylated ORF2 protein of the invention to promote multimerisation of ORF2, or to promote assembly of virus or VLP particles. In one embodiment, the use comprises adding a K411 acetylated ORF2 protein to a solution for assembly of VLP particles, wherein said solution comprises a non-acetylated ORF2 protein, wherein the efficiency of assembly of the VLP particles is increased compared to a control without K411 acetylated ORF2 protein.

HDAC6 inhibitors:

HDAC 6inhibitors herein include molecules that may reduce HDAC6 expression and/or activity in a cell. For example, an HDAC6inhibitor may be a molecule (referred to herein as a "HDAC 6 chemical inhibitor") that, when bound to or interact with an HDAC6 protein (e.g., SEQ ID No.5) or a functional fragment thereof, reduces or eliminates the biological activity of the protein, including the strength or duration of the biological activity. As another example, an HDAC6inhibitor may also be a nucleic acid molecule that prevents or reduces expression of a gene encoding HDAC6 protein, i.e., prevents or reduces gene transcription, mRNA maturation, mRNA translation, and post-translational modifications (referred to herein as an "HDAC 6 inhibitory nucleic acid molecule"). Thus, HDAC 6inhibitors would reduce or eliminate the deacetylation activity of HDAC6 protein deacetylase on the K411 acetylated lysine of ORF2 in cells.

HDAC6 inhibitory nucleic acid molecules

Examples of HDAC6 inhibitory nucleic acid molecules include, but are not limited to, for example, antisense nucleotide sequences specific for HDAC6 protein gene or mRNA sequences; a ribozyme specific for the HDAC6 protein mRNA; aptamers (aptamers) specific for HDAC6 protein mRNA; and interfering rna (rnai) specific for HDAC6 protein mRNA; or an expression construct thereof (any nucleic acid molecule capable of expressing an RNAi-producing molecule, including expression vectors). These inhibitory nucleic acid molecules can be constructed and generated by those skilled in the art of genetic engineering based on knowledge in the art about transgenes and inhibition of gene expression. (Clarke, A.R. (2002) Transmission technologies. principles and Protocols,2nd Ed. Humana Press, Cardiff University; US patent 20020128220. Gleeve, martin. TRPM-2antisense therapy; Puerta-Fer index E et al (2003) Ribozymes: vectors in parameters in details of RNA Protocols. FEMS Microbiology Reviews 27: 75-97; Kikuchi et al, 2003.RNA aptamers targeted to domains II of nanoparticles C viruses S channels bound to genes upper logic region J.Biochem.263, siRNA-270; Rege.A. Regulation A.2002. RNA signaling A.inner noise 3. Nature 22. inner noise).

In some embodiments, the HDAC6inhibitor is an RNAi molecule that directs the sequence-specific degradation of a histone acetylase HDAC6mRNA molecule by a process known as RNA interference.

RNAi approaches to inhibit target gene expression and target gene knockdown are well known in the art. RNAi includes, but is not limited to, siRNA and shRNA. sirnas are small or short interfering RNA molecules that can be transfected into cells using lipophilic transfection reagents to achieve transient knockdown of target genes. The siRNA may be, for example, a 19-25 nucleotide long double-stranded RNA molecule with a 2nt overhanging residue at its 3' end. In use, a single siRNA molecule directed to a single target site may be used, or a mixture of multiple, e.g., three, pairs of siRNA duplex molecules may be used. shRNA is a small hairpin and a short hairpin RNA. Nucleic acid plasmids encoding shRNA can be introduced into cells by lipopermeabilization transfection. The shRNA molecule expression and target gene inhibition can be achieved transiently or stably by using the shRNA. The shRNA plasmid may be composed of a mixture of one or more, e.g., 3-5, plasmids, each of which may, for example, contain a 19-25nt shRNA specific for the target gene and a 6bp stem-loop region. The shRNA-encoding nucleic acid can be placed under the control of, for example, a constitutive or inducible promoter. After transfection, cells stably expressing the shRNA can be selected. shRNA encoding plasmids can also be delivered into target cells by lentiviral particles for transient or stable knockdown of expression of target genes.

In one embodiment of the invention, the expression of the target gene HDAC6 is inhibited by introducing into the cell an siRNA synthesized in vitro. In another embodiment, inhibition of HDAC6 gene expression is achieved by constructing shRNA expression vectors in vitro and then transferring the vectors to cells for transcription to produce shRNA. In yet another embodiment, the shRNA expressing nucleic acid is constitutively expressed in the cell, thereby effecting constitutive knockdown of the target gene HDAC 6. In some embodiments, in the methods of HEV virus or virus-like particle or ORF2 production of the invention, the cells that are constitutively knocked down using HDAC 6. In yet another embodiment, the shRNA expressing nucleic acid is under the control of an inducible promoter, whereby shRNA expression and HDAC6 knock-down can be induced by an inducer, resulting in a conditionally knockdown cell of HDAC 6. In some embodiments, in the methods of HEV virus or virus-like particle or ORF2 production of the invention, conditional knockdown of cells using HDAC6, and preferably prior to infection with HEV virus or introduction or expression of ORF2 encoding nucleic acid, induces a decrease in the expression level of HDAC 6.

Thus, also contemplated in some embodiments of the invention are the use of HDAC6 knockdown cells, including HDAC6 constitutive knockdown cells or HDAC6 conditional knockdown cells for the methods of the invention. Herein, a cell with a constitutive knock-down of HDAC6 refers to a cell wherein the expression of HDAC6 is stably knocked down/inhibited, e.g., due to constitutive expression of inhibitory nucleic acid molecules, e.g., HDAC6RNAi molecules, in the cell. A cell with conditional knockdown of HDAC6 is a cell in which expression of HDAC6 is conditionally knocked down/inhibited, e.g., a cell in which expression level of HDAC6 is reduced in the presence of an inducer due to inducible expression of an inhibitory nucleic acid molecule, e.g., an RNAi molecule, that inhibits expression of HDAC6in the cell. Various methods are known in the art for generating conditional knockdown cells, see e.g., wangting et al, developments in conditional RNA interference technology research, advances in modern biomedicine, vol 15, No. 3, 2015, p 547-549; pfeiffenberger E et al, Conditional RNAi Using the Lentiviral GLTR System. methods Mol biol.2016; 1448:121-38(doi:10.1007/978-1-4939-3753-0_ 10); fewell GD et al, Vector-based RNAi aptamers for tables, inductor and gene-wide screens, Drug Discov today.2006 Nov; 975-82.Epub 2006Sep 26; US20130096370a 1.

In one embodiment, the invention relates to an siRNA molecule having RNAi activity against a target HDAC6RNA, wherein the siRNA molecule comprises a sequence complementary to an HDAC6RNA sequence. The nucleotide sequence of HDAC6 is known in the art. An exemplary HDAC6 nucleotide sequence (SEQ ID NO:6) is given in FIG. 7. Based on this sequence information, one skilled in the art can generate suitable HDAC6 inhibitory siRNA molecules using methods known in the art. The siRNA of the present invention may be unmodified or chemically modified. Chemical modifications to the siRNA can be used, for example, to increase the resistance of the siRNA to nuclease degradation in vivo and/or to improve the uptake of the siRNA by the cell.

There are examples in the art of sugar, base and phosphate modifications that can be introduced into nucleic acid molecules to enhance their nuclease-resistant stability and efficacy. For example, nuclease resistant groups, such as 2 'amino, 2' -C-allyl, 2 '-fluoro, 2' -O-methyl, etc., have been introduced into oligonucleotides to enhance their stability and/or to enhance their biological activity (see, e.g., Usman and Cedergren, 1992, TIBS.17, 34; Usman et al, 1994, Nucleic Acids Symp. Ser.31, 163; Burgin et al, 1996, Biochemistry, 35,14090). Carbohydrate modifications of nucleic acid molecules have also been widely described in the art (see Eckstein et al, PCT International publication WO 92/07065; Perrault et al, Nature, 1990,344,565,568; Pieken et al, Science, 1991, 253, 314-.

In some embodiments, the invention relates to shRNA molecules having RNAi activity against a target HDAC6 RNA. The shRNA molecules can be encoded by and expressed from a genome-integrated transgene or a plasmid-based expression vector. Thus, in some embodiments, the molecule capable of inhibiting mRNA expression is a transgene or a plasmid-based expression vector encoding a small interfering nucleic acid. Such transgenes and expression vectors may use polymerase II or polymerase III promoters to drive expression of these shrnas and result in functional sirnas in cells. Inducible and tissue-specific expression systems can also be used to drive expression of these shrnas. In some embodiments, the transgene and the expression vector are under the control of a tissue-specific promoter. In other embodiments, the transgene and expression vector are controlled by an inducible promoter, such as a tetracycline-inducible expression system. A variety of carriers for shRNA delivery are known in the art, including viral carriers, such as Lentiviral particles (see, e.g., review Manjunath N et al, Lentiviral delivery of short hairpin RNAs, Adv Drug Deliv Rev.2009Jul 25; 61(9):732-45.doi:10.1016/j. addr.2009.03.004.epub 2009Mar 31).

In addition to siRNA and shRNA molecules, other RNAi molecules, such as dsRNA, can also be chemically synthesized or recombinantly produced. Methods of designing RNAi molecules and methods of transfecting RNAi molecules into cells and animals are well known in the art and are commercially available (Verma n.k. et al, j.clin.pharm. ther.,28(5):395-404(2004), Mello c.c. et al, Nature,431(7006)338-42(2004), Dykxhoorn d.m. et al, nat. rev.mol. cell. biol.4(6):457-67(2003), Santa cruz biotechnology (Dallas, USA)).

In addition to RNAi, other inhibitory nucleic acid molecules that may be used include antisense nucleic acids, ribozymes, aptamers (aptamers), and the like. Antisense and ribozyme inhibition strategies work by reducing the expression of gene products or by cleaving transcripts specifically (Carter and Lemoine Br. J. cancer.67(5): 869-.

HDAC6 chemical inhibitors

The chemical inhibitor of HDAC suitable for use in the methods of the present invention may be any HDAC6inhibitor known in the art, including a selective HDAC6inhibitor and a non-specific HDAC inhibitor that inhibits HDAC6 and other HDAC enzymes, such as TSA, but is preferably an HDAC6 selective inhibitor, such as TBSA.

Some selective HDAC 6inhibitors may have nanomolar or even picomolar HDAC6 inhibitory activity and exhibit significant HDAC6 selectivity relative to other HDAC enzymes. As used herein, "HDAC 6 selectivity" means that a compound binds to HDAC6 to a significantly greater extent, e.g., 10, 50, 100, 500 or more fold greater, than any other type of HDAC enzyme, e.g., HDAC 1or HDAC2, i.e., the compound is selective for HDAC6 over any other type of HDAC enzyme. Selective inhibition of HDAC6 relative to other HDAC isoforms may reduce toxicity or side effects on cells or tissues.

Various HDAC 6inhibitors have been studied. See, for example, Butler et al, "Rational Design and Simple Chemistry Yield a Superior, Neuroprotective HDAC6Inhibitor, Tubastatin A," J Am Chem Soc 2010,132 (31): 10842-10846; kalin et al, "Second-Generation Histone deacylase 6Inhibitors enhancement of Foxp3+ T-Regulatory Cells," J Med Chem 2012,55(2): 639-; west and Johnstone, j.clin.invest.2014,124, 30-39; mottamal et al, molecules 2015,20, 3898-. Batchus et al provide a detailed discussion of HDAC 6inhibitors (Clinical Science (2016)130, 987-. See also WO2018039581a 1; WO2018034801a 1; sabrina Dallavalle, Claudio Pisano, and Franco Zunino, "Development and therapeutic impact of HDAC6-selective inhibitors," Biochemical Pharmacology,84(6), (2012), p 756-76. All of the above documents are incorporated by reference herein in their entirety.

Non-limiting examples of HDAC 6inhibitors include, rocilinostat (ACY-1215), ACY-241, Tubacin, Tubastatin A (TBSA), CAY10603, Nexturastat A, HPOB, ACY-738(N-hydroxy-2- (1-phenylcyclopropyamino) pyrimide-5-carboxamide), ACY-775(2- ((1- (3-fluorophenyl) cyclohexoxy) amino) -N-hydroxypyramide-5-carboxamide), and ACY-1083. Other HDAC 6inhibitors include Vorinostat, LBH589, ITF2357, PXD-101, Trichostatin A (TSA). These inhibitors are commercially available.

In one embodiment, the HDAC6inhibitor of the present invention is an HDAC6 selective inhibitor selected from the group consisting of:

Rocilinostat(ACY-1215)

Citarinostat(ACY-241)

CAY10603

Tubastatin A

Tubac in

ACY-1083

or a pharmaceutically acceptable salt thereof.

In one embodiment, the HDAC6inhibitor is TSA. In one embodiment, the HDAC6inhibitor is TBSA. In another embodiment, the HDAC6inhibitor is CAY 10603.

HDAC6 activators

HDAC6 activators herein include molecules that can increase HDAC6 expression and/or activity in a cell. For example, an HDAC6 activator may be a molecule that, when bound to or interacting with an HDAC6 protein (e.g., SEQ ID No.5) or a functional fragment thereof, increases the strength or prolongs the duration of the biological activity of said protein. HDAC6 activators may also be those compounds that allow for increased expression of the gene encoding HDAC6 protein.

The present invention relates, in one aspect, to the use of HDAC6 activators for the development of medicaments or therapeutic compositions for the prevention and treatment of HEV viral infection. In one embodiment, the compound consists of a genetic construct comprising a nucleotide sequence encoding HDAC6 (e.g. SEQ ID No.6) which allows expression of HDAC6 protein (e.g. SEQ ID No.5) in a mammalian cell, e.g. a human or non-human mammalian cell, preferably in a hepatocyte.

In one embodiment, the present invention provides a method of treating HEV infection using an HDAC6 activator, wherein the HDAC6 activator is a genetic vector or expression vector capable of expressing a peptide or protein capable of mimicking or reproducing the biological activity of an HDAC6 protein deacetylase in a mammalian cell, preferably a hepatocyte, said vector comprising a nucleotide sequence selected from the group consisting of:

a) nucleotide sequence encoding HDAC6 protein

b) a) a fragment of a nucleotide sequence, and

c) sequences homologous to a) and b).

Homologous sequences may be constructed, for example, by introducing conservative or non-conservative substitutions, including the insertion of one or more nucleotides, the addition of one or more nucleotides at any end of the molecule, or the deletion of one or more nucleotides at any end or within the sequence, wherein the homologous sequence expresses a protein having the deacetylase activity of the HDAC6 protein which retains the deacetylation of acetylated lysine at position K411 of ORF2 protein.

In some embodiments, the homologous sequences have a degree of identity of at least 80%, preferably at least 85%, or more preferably at least 95%. In other embodiments, the homologous sequence is a variant of the reference sequence, e.g., a variant with 1-10 amino acid changes, particularly conservative amino acid changes.

As previously described, these constructs and vectors can be constructed by those skilled in the art according to conventional methods (Sambrook et al, Molecular Cloning; A Laboratory Manual, second edition, Cold spring harbor Press, Cold spring harbor, New York (1989)).

Screening method

In another aspect of the invention, the invention also provides methods of identifying HDAC6 modulators (including inhibitors and activators) and assessing their activation and/or inhibition of ORF2 acetylation and HEV viral replication, comprising the steps of:

i) contacting a cell comprising a nucleic acid encoding ORF2 with a potential compound and incubating under suitable conditions,

ii) determining the level of acetylation of ORF2 in the cells of i), or a parameter indicative of viral replication or assembly, and

iii) identifying the compound as an inhibitor or activator of HDAC6 protein activity when a change in a parameter indicative of an increase in the level of acetylation of the ORF2 protein and/or indicative of an increase in viral replication is observed, or when a change in a parameter indicative of a decrease in the level of acetylation of ORF2 and/or a decrease in viral replication is observed.

The level of acetylation of ORF2 protein can be detected using methods known in the art, such as immunoblotting or western blotting, or the methods exemplified in the examples. The level of viral replication can be detected using methods known in the art, or as exemplified in the examples.

The invention also includes the use of the HDAC6 modulators obtained by the screening for use in the methods and compositions of the invention.

Production method and cell culture System of the present invention

Based on the foregoing findings, the present inventors propose methods for improving the production of HEV virus, virus-like particle, or ORF2 protein (which may be used in formulating a HEV virus vaccine) by modulating HDAC6 expression and/or activity. The inventors also propose a cell culture system for said production. The HEV virus, virus-like particle or ORF2 protein produced by the methods and culture systems of the invention can be used to formulate anti-HEV viral medicaments, such as immunological compositions or vaccines.

Cell culture system

In one aspect, the invention provides a cell culture system for HEV virus, virus-like particle, or ORF2 protein production, the system comprising: (i) a cell comprising a nucleic acid encoding an ORF2 polypeptide; and (ii) an HDAC6 inhibitor. In one embodiment, the inhibitor is an HDAC6 inhibitory nucleic acid molecule, particularly an RNAi such as an siRNA or shRNA or expression construct thereof, introduced into the cell. In one embodiment, the inhibitor is an HDAC6 chemical inhibitor, particularly an HDAC6 selective inhibitor, added to a culture of said cells. In one embodiment, the cell is a HEV virus-infected cell. In another embodiment, the cell is a recombinant cell into which an ORF2 expression construct has been introduced. In one embodiment, HDAC6 expression in the cell is knocked down by HDAC6 inhibitory nucleic acid molecules prior to infection of the cell with HEV virus or prior to expression of ORF2 by the cell. The cell culture system of the present invention can be used for the production method of the present invention.

HEV virus production method

In one aspect, the invention provides methods for improving HEV virus production in cultured cells in vitro of HEV virus. In one embodiment, the method of the invention comprises: HEV infected cells are cultured in vitro in the presence of an HDAC6 inhibitor. In another embodiment, the method of the invention comprises: HDAC6 knockdown cells were infected with HEV and cultured in vitro. In another embodiment, the method of the invention comprises: HEV infected cells were treated with HDAC6 chemical inhibitors. In yet another embodiment, the invention includes harvesting HEV viral particles. In a further embodiment, the harvested HEV viral particles are formulated as a vaccine or an immunological composition. In yet another aspect, the invention also relates to the use of the harvested HEV viral particles or formulated vaccines or immunological compositions in the treatment of HEV infection.

As will be appreciated by those skilled in the art, any cell culture that allows replication of HEV virus (i.e., cells susceptible to HEV virus) can be used in the methods of the invention. Cell lines that can support HEV replication following viral infection are known in the art and include, but are not limited to, a549 (human lung cell carcinoma) and PLC/PRF/5 (human hepatocellular carcinoma). In addition, the porcine kidney epithelial cell line LLC-PK1 has also been shown to support HEV strain replication. Furthermore, the production of high titer hepatitis E virus in cell culture medium using HepG2(ATCC No. HB-8065) or HepG2/C3A (ATCC No. CRL-10741) has also been reported (see CN 106367397A).

In some embodiments of the invention, the cell used for HEV virus production is a mammalian cell, e.g., a human or porcine cell line, e.g., a liver or lung or kidney cell line, such as a PLC/PRF/5 human hepatoma cell or A549 human lung cell or HepG2/C3A human hepatoma cell line or LLC-PK1 porcine kidney cell line. In other embodiments of the present invention, it is preferred that the cells are A549 cells or PLC/PRF/5 cells.

In some embodiments, viral replication is increased in cells treated with the HDAC6inhibitor relative to cells not treated with the HDAC6 inhibitor. Increased viral replication refers to the production of greater amounts of HEV virus by cells treated with an HDAC6inhibitor compared to control cells not treated with an HDAC6inhibitor when HEV virus infects cells, e.g., human liver or lung cell lines, such as PLC/PRF/5 hepatoma cells or a549 lung cells. The level of viral replication can be determined, for example, by assaying for HEV antigen levels in an ELISA assay, e.g., by treatment with HDAC6 for a period of time after viral infection of cells, followed by collection of cell culture supernatants. In addition, the production of ORF2 can be measured to assess the level of virus production using methods well known in the art, e.g., the amount of ORF2 in cell supernatants and/or cell lysates can be detected using flow cytometry or fluorescence microscopy or immunoblotting, among other methods.

In some preferred embodiments, the present invention provides a method for the production of a medicament (e.g. an immunogenic composition or vaccine) comprising a virus, the method being based on in vitro cell culture of the virus, the method comprising: after infecting the cells with HEV virus and allowing sufficient time for the virus to replicate and produce in the cells, the virus is purified from the cells. In one embodiment, the method comprises: HDAC6 knockdown cells were infected with HEV virus and the infected cells were cultured. In yet another embodiment, the method comprises: the HDAC6 chemical inhibitor may be added to the virus at the same time the virus is produced on the cell, or may be added at a different stage of virus production after infection. Preferably, after viral infection of the cells, e.g., about 3 weeks, the cells are treated with the HDAC6 chemical inhibitor (e.g., for 3 days).

In some preferred embodiments, the present invention provides a method of improving HEV virus production, comprising: in cells for in vitro HEV virus culture, the cells are modulated for HDAC6 expression and/or activity. Preferably, the cells are mammalian cells, preferably human or porcine cell lines, in particular a549 lung cells and PLC/PRF/5 liver cancer cells. Preferably the method comprises: culturing the cell in the presence of an HDAC6inhibitor, wherein viral replication is increased in the cell relative to a cell not treated with an HDAC6 inhibitor. Preferably, the method comprises the steps of: infecting the cells with HEV virus and optionally harvesting the HEV virus.

In a preferred embodiment, the method comprises: (i) introducing an HDAC6RNAi molecule (particularly siRNA or shRNA) or a nucleic acid encoding same into a cell to generate an HDAC6 knockdown cell, (ii) infecting the HDAC6 knockdown cell with HEV virus; (iii) culturing the cells for a period of time and optionally harvesting the virus.

In a preferred embodiment, the method comprises: (i) infecting the cells with HEV virus; (ii) (ii) applying a chemical inhibitor of HDAC6, e.g. a HDAC6 selective inhibitor, to the cells, (iii) culturing the cells for a period of time and optionally harvesting the virus.

Preferably, the method further comprises: the harvested virus is formulated into an immunological composition or vaccine for treating or preventing HEV infection.

Method for producing virus-like particles

In one aspect, the invention provides a method of improving HEV Virus Like Particle (VLP) production, comprising: inhibiting HDAC6 expression and/or activity in a cell comprising a nucleic acid encoding an ORF2 polypeptide. Preferably, the assembly of HEV VLPs and/or VLP production in a cell can be improved by the methods of the invention. The virus-like particles obtained by the method of the invention can be used for preparing medicaments against HEV virus infection, such as immunogenic compositions or vaccines.

Without being bound by theory, it is believed that inhibiting HDAC6 deacetylase activity in cells may increase the acetylation level of ORF2 at K411; acetylated ORF2 protein has higher resistance to proteasomal degradation and the ability to promote dimerization and multimerization of ORF2 to form VLPs compared to the non-acetylated ORF2 protein. Without being bound by theory, it is believed that: by inhibiting HDAC6 treatment, the amount of ORF2 in the cells can be increased and ORF2 present in free form can be decreased, increasing the efficiency of cellular expressed ORF2 assembly into VLPs.

Thus, in one embodiment, the invention relates to a method of promoting VLP assembly efficiency by increasing the amount of acetylated ORF2 in a cell. In one embodiment, the method of the invention comprises: culturing cells comprising a nucleic acid encoding an HEV ORF2 polypeptide in vitro in the presence of an HDAC6 chemical inhibitor. In another embodiment, the method of the invention comprises: a nucleic acid encoding the ORF2 polypeptide was transferred into HDAC6 knockdown cells and the cells were cultured in vitro. In the methods of the invention, in some embodiments, the cell is a cell that itself has an acetylation-deacetylation system, e.g., a mammalian cell, an insect cell, preferably an insect cell, especially an sf9 cell.

It is known in the art that ORF2 structural proteins of HEV can be obtained by various expression systems, and then assembled into virus-like particles (VLPs) in vivo or in vitro, thereby obtaining virus-like particles of HEV. For example, it is known in the art that baculovirus/insect cells can be used to express fragments of HEV ORF2 (e.g., aa112-aa607 or aa14-aa608 or aa112-660), and self-assembled HEV VLPs can be obtained in cell lysates or in cell supernatants. Furthermore, it has been demonstrated that the insertion of the 11 amino acid B-cell epitope tag at the C-terminus of truncated ORF2 results in a chimeric ORF2 capsid protein that can still form icosahedral particles. The chimeric HEV VLPs stimulate an immune response against the inserted epitope after oral administration (Niikura et al, 2002.Virology 293,273- "280). These expression systems and ORF fragments are suitable for use in the present invention.

In some embodiments, expression systems for the production of virus-like particles of the invention include, but are not limited to, expression systems using insect cells and mammalian cells. Preferred expression systems include baculovirus expression systems using insect cells. Methods for the treatment and preparation of baculovirus vectors and baculovirus DNA, and insect cell culture, are known in the art. See, e.g., A Manual of Methods for Baculoviral Vectors and Instrument Cell Culture Procedures. For example, HEV capsid protein ORF2 or a truncated fragment thereof can be cloned into a baculovirus vector for infection of a suitable host cell. (see, e.g., O' Reilly et al, "Bacillus Expression Vectors: A Lab Manual," Freeman & Co. 1992). Insect cell lines (e.g., sf9 or Tn5) can be transformed with a transfer vector comprising a polynucleotide encoding an ORF2 polypeptide of the invention. Transfer vectors include, for example, linearized baculovirus DNA and plasmids containing the desired polynucleotide. Host cell lines can be co-transfected with linearized baculovirus DNA and plasmids to produce recombinant baculovirus.

In a preferred embodiment, the present invention uses a baculovirus/insect cell expression system for VLP production. In a further embodiment, VLP production is performed using a truncated ORF2 fragment, said ORF2 fragment preferably comprising an aa125-601, aa112-aa607, or aa112-660 sequence, but lacking the amino acid sequence 1-111 of the N-terminus. In one embodiment, the ORF2 fragment is fused at the C-terminus to a peptide antigen, such as a B-cell epitope tag.

Methods for producing and purifying virus-like particles are known in the art (see, e.g., Expression and self-assembly of Expression virus-like particles of hepatitis E virus. Li TC, Yamakawa Y, Suzuki K, Tatsumi M, Razak MA, Uchida T, Takeda N, Miyamura T., J Virus. 1997 Oct; 71(10):7207-13. Expression elements of the peptide protein for Expression-assembly virus-like particles of hepatitis E virus. Li TC, Takeda N, Miyamura T, Matsuura Y, Wang JC, Engval H, Hammar L, Xylag, chemistry J. Virol. 200300. RH; Yakawa N, Miyamura T, Matsuura Y, Wang JC, Engval H, Hammar L, Xyland, chemical J. Virol. RH. 200300-10. 9. mu. 9. acquisition: 14. 9. nucleic acids of nucleic acids, III et 99, 5. 9. nucleic acids). These documents are incorporated herein by reference.

In some preferred embodiments, the present invention provides a method of improving the production of HEV virus-like particles, comprising: modulating HDAC6 expression and/or activity of a cell in a cell comprising a nucleic acid encoding ORF2 or a fragment thereof. In a preferred embodiment, the cell is an insect cell, a mammalian cell, especially an insect cell, such as sf9 cell. In a preferred embodiment, the fragment of ORF2 is a fragment of ORF2 capable of self-assembly into VLPs, preferably a fragment of ORF2 truncated by the N-terminal aa1-111 sequence; preferably the ORF2 fragment comprises aa125-601 or 125-607 amino acid residues, more preferably the ORF2 fragment is an aa112-660 fragment. In one embodiment, the ORF2 fragment is fused at the C-terminus to an amino acid antigenic peptide, such as a B cell epitope tag. In a preferred embodiment, the method comprises: culturing the cell in the presence of an HDAC6inhibitor, wherein assembly and/or production of VLPs in the cell is increased relative to a cell not treated with an HDAC6 inhibitor. In a preferred embodiment, the method comprises: (i) introducing an HDAC6RNAi molecule (particularly siRNA or shRNA) or a nucleic acid encoding same into a cell, resulting in an HDAC6 knockdown cell, (ii) introducing a nucleic acid encoding ORF2 or fragment thereof into said HDAC6 knockdown cell; (iii) culturing the cells for a period of time and optionally harvesting the virus-like particles. In a preferred embodiment, the method comprises: (i) introducing into a cell a nucleic acid encoding ORF2 or a fragment thereof; (ii) (ii) applying a chemical inhibitor of HDAC6, e.g. a selective inhibitor of HDAC6, to the cells, (iii) culturing the cells for a period of time and optionally harvesting the virus-like particles. Preferably, the method further comprises: the harvested virus-like particles are formulated into an immunological composition or vaccine for treating or preventing HEV infection.

Method for producing ORF2 polypeptide

In yet another aspect, the invention provides a method of improving HEV ORF2 polypeptide production, comprising: in a cell comprising a nucleic acid encoding an HEV ORF2 polypeptide, the cell is modulated for HDAC6 expression and/or activity. Preferably, the cells are insect cells such as sf9 cells, mammalian cells such as 293T cells and HeLa cells. Preferably, the cell is an HEV ORF2 transgenic cell, or an HEV virus-infected cell.

In one embodiment, the ORF2 polypeptide can be any ORF2 full-length sequence or truncated fragment and homologs or variants thereof as described previously herein. Preferably, the ORF2 fragment is an ORF2 fragment truncated by the N-terminal aa1-111 sequence; preferably the ORF2 fragment comprises aa125-607 amino acid residues, more preferably the ORF2 fragment is an aa112-660 fragment. Preferably, the fragment retains the characteristics of K411 acetylation and deacetylation by HDAC6, and retains the ability to self-assemble to form viral capsids or virus-like particles, preferably immunogenic.

In a preferred embodiment, the method comprises: culturing the cell in the presence of an HDAC6inhibitor, wherein the stability of ORF2 in the cell is increased, and preferably the amount of ORF2 in the cell is increased, more preferably the amount of ORF2 multimers in the cell is increased, relative to a cell not treated with the HDAC6 inhibitor.

In a preferred embodiment, the method comprises: (i) introducing into a cell an HDAC6RNAi molecule (in particular an siRNA or shRNA) or a nucleic acid encoding said RNAi molecule, resulting in an HDAC6 knockdown cell, (ii) introducing into said HDAC6 knockdown cell a nucleic acid encoding ORF2 or a fragment thereof; (iii) culturing the cells for a period of time and optionally harvesting the ORF2 produced. In a preferred embodiment, the method comprises: (i) introducing into a cell a nucleic acid encoding ORF2 or a fragment thereof; (ii) (ii) applying to the cells a chemical inhibitor of HDAC6, e.g. a selective inhibitor of HDAC6, (iii) culturing the cells for a period of time and optionally harvesting the resulting ORF 2. ORF2 can be harvested from cell supernatants and/or cell lysates. Preferably, the method further comprises: harvested ORF2 is formulated into an immunogenic composition or vaccine for treating or preventing HEV infection.

Use of HDAC 6inhibitors

In one aspect, the invention provides the use of an HDAC6inhibitor, such as an HDAC6 inhibitory nucleic acid or HDAC6 chemical inhibitor, (a) for increasing the K411 acetylation level of HEV ORF2, (b) for increasing the stability and/or ORF2 yield of HEV ORF2, (c) for increasing HEV virus replication and/or virus yield, or (d) for increasing the assembly of HEV VLPs and/or VLP yield. Preferably, the application comprises: HEV infected cells or cells introduced a recombinant ORF2 expression construct are cultured in the presence of an HDAC6 inhibitor.

In yet another aspect, the invention provides a cell culture comprising an HDAC6 knockdown cell, wherein the cell comprises a nucleic acid encoding HEV ORF 2. Preferably, the cell is a HEV-infected cell, or a cell into which a recombinant ORF2 expression construct has been introduced. The cell culture can be used to produce HEV virus, virus-like particle, or ORF2 polypeptide.

In any of the above-described methods of production and use of HDAC 6inhibitors of the invention, in a preferred embodiment, an HDAC6inhibitor is used to knock down the expression level of HDAC6in a cell, preferably using HDAC6siRNA or shRNA. In yet another embodiment, the HDAC6 knockdown cell is a cell comprising an siRNA or shRNA expressing nucleic acid. In one embodiment, the cell is a constitutively knocked-down cell for HDAC6, wherein the siRNA or shRNA expressing nucleic acid is constitutively expressed in the cell. In yet another embodiment, the cell is a cell with conditional knockdown of HDAC6, wherein the siRNA or shRNA expressing nucleic acid is under the control of an inducible promoter and expresses and reduces HDAC6 activity in the presence of an inducer. In one embodiment, the siRNA or shRNA expressing nucleic acid is stably integrated in the genome of the cell.

In any of the above described methods of production and use of HDAC 6inhibitors of the invention, in another preferred embodiment, an HDAC6inhibitor is used to reduce the activity of HDAC6in a cell, wherein said HDAC6inhibitor is a chemical inhibitor, e.g., TSA, TBSA or CAY10603, especially TBSA. In one embodiment, the concentration of Trichostatin A (TSA) is 0.2. mu.M to 5. mu.M, e.g., 0.5. mu.M, 1. mu.M, 2. mu.M, 5. mu.M. In one embodiment, the concentration of Tubastatin A (TBSA) is 1. mu.M-10. mu.M, e.g., 3. mu.M, 6. mu.M, 10. mu.M. In one embodiment, the concentration of CAY10603 is between 0.1. mu.M and 1. mu.M, e.g., 0.2. mu.M, 0.5. mu.M, 1. mu.M. Preferred concentrations are TSA, 1. mu.M, TBSA, 6. mu.M, or CAY10603, 0.2. mu.M.

In any of the above described methods of production and use of HDAC 6inhibitors of the invention, in one embodiment, the virus or virus-like particle or ORF2 is harvested by harvesting the cell culture medium after a period of HDAC6inhibitor treatment; and/or harvesting the virus or virus-like particle or ORF2 by lysing the cells. Optionally, the harvested virus or viral particle or ORF2 may be purified. The purification may comprise at least one step selected from: clarification, ultrafiltration/diafiltration, ultracentrifugation (e.g. density ultracentrifugation, in particular sucrose gradient density ultracentrifugation) and chromatography or any combination thereof. The above steps may be combined in any manner depending on the desired level of purity.

In some preferred embodiments, by inhibiting HDAC6 expression and/or activity in a cell, the K411 acetylation level of HEV ORF2 protein is increased, and/or the stability of ORF2 in a cell is increased and/or the amount of ORF2 in a cell is increased, particularly the amount of ORF2 multimer in a cell is increased. In some preferred embodiments, the formation of viral inclusion bodies in the cell is increased by inhibiting HDAC6 expression and/or activity in the cell. In some preferred embodiments, the replication of HEV virus in the cell is increased by inhibiting HDAC6 expression and/or activity in the cell. In some preferred embodiments, the assembly of VLPs and/or the production of VLPs in a cell is produced by inhibiting HDAC6 expression and/or activity in the cell.

The hepatitis E virus, ORF2 protein and virus-like particles produced using the methods of the invention can be purified or partially purified by methods known to those skilled in the art from the cell or culture supernatant from which they are produced, before use as immunogens in the pharmaceutical compositions and vaccines of the invention. The invention therefore also relates to the use of the HEV acetylation ORF2 protein, viruses and virus-like particles produced from the invention for the preparation of immunological compositions and vaccines. For example, the virus may be formulated as a live vaccine or an inactivated vaccine (e.g., a formalin inactivated vaccine) to prevent or treat hepatitis e in a mammal. For example, acetylated ORF2 protein or virus-like particle may be formulated in an immunological composition comprising an adjuvant for the prevention or treatment of hepatitis E in a mammal. Alternatively, the immunogen may be a partially or substantially purified recombinant protein or virus-like particle.

Method of treatment

In yet another aspect, the invention also provides a method of treating HEV viral infection comprising administering to an individual infected with HEV virus a modulator of acetylation of HEV ORF2 protein K411, wherein the modulator reduces the level of acetylation of ORF2 protein K411. Preferably, the modulator increases the expression and/or activity of HDAC6in a cell, preferably the modulator is an HDAC6 expression nucleic acid. Preferably, the subject is a mammal, such as a human or a pig.

In any of the foregoing aspects of the invention, preferably, the HEV virus is a HEV virus of genotypes 1-4, preferably genotypes 1 and 4.

In any of the foregoing aspects of the invention, preferably, the HEV ORF2 polypeptide comprises a sequence selected from:

(a) the full-length sequence of HEV ORF2 protein of genotypes 1-4, preferably genotypes 1 and 4;

(b) a truncated fragment of (a), said fragment comprising the peptide sequence E (P/L) TVK⁴¹¹LYTSVEN peptide sequences, preferably comprising the sequence aa125-601, especially aa125-607, particularly preferably consisting of the sequence aa 112-660;

(c) a homologue or variant of (a) or (b).

These and other aspects and embodiments of the invention are described in the accompanying drawings (brief description of the drawings follows) and in the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the invention. The following examples further illustrate the invention, however, it is to be understood that the examples are described by way of illustration and not limitation, and that various modifications may be made by those skilled in the art.

Examples

The following abbreviations used in the examples and figures have the following meanings:

ORF2 represents the ORF2 polypeptide of HEV virus, including the full-length sequence or a truncated fragment thereof;

ORF2(112-660) represents the truncated fragment aa112-660 of ORF2 of HEV;

numbers

1 and 4, e.g., 1-ORF2 or 4-ORF2, preceding ORF2 indicate that the ORF2 polypeptide is from HEV genotype 1or genotype 4.

Addition of a GFP or FLAG, e.g., "GFP-ORF 2" or "FLAG-ORF 2", before ORF2 indicates an ORF2 polypeptide or fragment with a GFP or FLAG tag at the N-terminus.

K411R after ORF2, e.g., ORF2^K411ROr ORF2^K411R(112-660) indicating that the ORF2 polypeptide or fragment has a lysine to arginine mutation at position K411. Thus, for example, 1-ORF2^K411R(112-660) represents a nonacetylated mutant of the truncated fragment aa112-660 of HEV genotype 1ORF2, in which the lysine at position K411 is mutated to arginine; and 4-ORF2^K411R(112-660) represents a nonacetylated mutant of a truncated fragment of HEV genotype 4 ORF2 in which lysine at K411 was mutated to arginine.

TSA: HDAC6inhibitor Trichostatin a;

TBSA: the HDAC6inhibitor Tubastatin a;

CAY: the HDAC6inhibitor CAY 10603;

anti-Acetyl-Lys anti-acetylated lysine antibody;

IP: performing immunoprecipitation;

IB: immunoblotting;

in the examples and figures, reference to ORF2 refers to the aa112-660 fragment of ORF2, unless it is specifically stated that the ORF2 polypeptide used is a full-length protein.

General methods and materials

Plasmid construction

The full length and 112-660aa fragment of 1-ORF2 (GenBank accession No.: JQ655734) were PCR amplified from HEV genotype 1 strain W2-1. The full length and 112-660aa fragment of 4-ORF2 (GenBank accession No.: JQ655736) were PCR amplified from HEV genotype 4 strain W2-5. The amplified fragment was cloned into pAcGFP1-C vector (Clontech, Mountain View, CA). To generate HA-tagged ORF2, HEV ORF2 was cloned in pCMV-HA (clontech). HDAC6 was cloned from HEK293T cDNA by PCR and inserted into pCMV-flag (Clontech). Site mutagenesis using KOD Neo DNA polymer and the following primers produced mutants that were not acetylated:

primer GCGAGCCGACTGTTAGGCTGTATACATCTG for 1-ORF2^K411R,

Primer CGAGCCGACAGTACGACTTTACACCTCAGT for 4-ORF2^K411R。

Cell culture, drug treatment and plasmid transfection

HeLa (American type culture Collection [ ATCC ]]CCL-2), HEK293T (ATCC, CRL-1573) and A549(ATCC, CCL-185) cells at 37 ℃ and 5% CO₂In DMEM (Hyclone, Logan, UT) with 10% fetal bovine serum, 100U/ml penicillin and 100. mu.g/ml streptomycin. To enrich for acetylated proteins, HEK293T cells, HeLa cells and PLC/PRF5 cells (ATCC, CRL-8024) were treated with 0.5 μ M Trichostatin A (Sigma, St. Louis, MO.) or 3 μ M Tubastatin A (Selleck, Houston, TX, Cat #: S8049) for 8 hours. Sf9 cells were treated with 1. mu.M Trichostatin A or 6. mu.M Tubastatin A or 0.2. mu.M CAY10603(Selleck Cat #: S7596) for 5 days. For transfection, 293T cells were grown to approximately 70% confluence using polyEthyleneimine (PEI, Alfa Aesar, Cat #:43896) was plasmid transfected. HeLa cells or A549 cells were transfected with lipofectamine 2000(Life Technologies, Grand Island, NY) according to the protocol provided by the manufacturer.

Antibodies and immunofluorescence

Rabbit anti-HDAC 6 polyclonal antibody (Cat #:12834-1-AP) and mouse anti-GAPDH monoclonal antibody (Cat #:60004-1-Ig, Clone No.:1E6D9) were purchased from Proteintetech (Proteintetech Group, Rosemont, IL). Mouse monoclonal antibodies to Flag (Cat #: F3165, Clone No.: M2) and a-tubulin (Cat #: T9026, Clone No.: DM1A) were purchased from sigma. Rabbit anti-acetylated lysine polyclonal antibody (Cat #:9441) was purchased from Cell signal Technology (Cell Signaling Technology, Boston, MA). Mouse anti-GFP antibodies (Cat #: AI10151) were purchased from Abgent (Abgent, San Diego, Calif.) and rabbit anti-GFP polyclonal antibodies were self-made in the laboratory. All fluorochrome-conjugated secondary antibodies were purchased from Invitrogen.

Cells cultured on coverslips were fixed with 10% TCA in PBS for 15 minutes at room temperature and permeabilized in 0.1% Triton X-100 in PBS buffer for 5 minutes. Cells were incubated with primary antibody diluted (1:100) in PBS with 3% BSA at 37 ℃ for 1 hour, or at 4 ℃ overnight. Then, cells were washed 3 times in PBS and incubated with fluorochrome-conjugated secondary antibodies diluted 1:500 in PBS with 3% BSA for 1 hour at 37 ℃. After washing the cells 3 times with PBS, the cells were mounted on slides with mowiol mounting medium (Sigma) containing DAPI for DNA staining. Live cell imaging data were acquired by UltraView VoX systerm (PerkinElmer, Waltham, MA). For microscopic observation of the fixed sample, images were taken by a Zeiss LSM710 microscope.

Immunoprecipitation (IP)

In lysis buffer (20mM Tris-HCl, pH 7.5,150mM NaCl,10mM NaVO) with 0.5% NP40₃10mM NaF and 0.5mM EGTA) were lysed on ice and the supernatant was collected by centrifugation at 4 ℃. anti-GFP or anti-Flag antibodies were incubated with protein A beads (GE Healthcare, Fairfield, CT) for 1 hour at 4 ℃. The beads were isolated, washed, and incubated with cell lysates for 2 hours at 4 ℃ and then washed 4 times with lysis buffer. Finally, the proteins on the beads are eluted using a sample buffer, andanalysis was by western blot.

RNAi assay

After growing the cells for 24 hours, they were transfected with siRNA to HDAC6 (Santa cruz biotechnology, Dallas, TX, Cat #: sc-35544) using lipofectamine 2000 according to the protocol described by the manufacturer. After 72 hours of transfection, cells were harvested for western blotting.

Mass spectrometric analysis

Cells were transfected with GFP-1-ORF2 or GFP-4-ORF2 plasmids for 24 hours and lysed in cell lysis buffer on ice for 20 minutes. Cell lysates were incubated at 4 ℃ for 2 hours and mixed with 20. mu.l protein A-Sepharose beads and rabbit anti-GFP antibody. After washing the beads with lysis buffer, the GFP-ORF2 polypeptide was resuspended in sample buffer. The samples were purified by SDS-PAGE separation and analyzed by acetyl-MS using gel sections.

HEV cultured in PLC/PRF/5 cells

PLC/PRF/5 cells were grown in minimal essential medium (MEM; Hyclone) supplemented with 10% Fetal Bovine Serum (FBS),100U/ml penicillin and 100. mu.g/ml streptomycin at 37 ℃ and 5% CO 2. Hepatitis e virus (virus accession number:AJ272108genotype 4) were added to PLC/PRF/5 cells with 2% FBS and culture supernatant was collected every three days (45).

Quantification of HEV ORF2 polypeptide by ELISA

HEV in the supernatant of the PLC/PRF/5 cell culture infected with HEV and ORF2 (112) -660) protein expressed from sf9 cell (ATCC CRL-1711) were diluted and tested by HEV antigen enzyme-linked immunosorbent assay (ELISA) (Wantai, Beijing, China) (45).

Observation with an electron microscope

Cultured 293T cells were fixed with 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer and then post-fixed with 1% osmium oxide in the same buffer. After dehydration with graded ethanol and displacement to propylene oxide, the samples were embedded in Pon 812 resin. The ultrathin sections were post-stained with uranyl acetate and lead citrate. Observations were made using a JEM-1400 Transmission Electron microscope (JEOL, Japan) and a specific imaging system for EM (830.10U3CCD camera, Gatan, USA).

Example 1: HEV capsid protein ORF2 is acetylated at conserved amino acid residue K411.

The N-terminus of ORF2 is not essential for its antigenicity and viral particle assembly (39). ORF2(112-660) shares the same biological properties as the wild-type virus (40). Thus, using this truncated ORF2, the interaction of HEV ORF2 with host proteins was studied and a number of molecules interacting with ORF2 were successfully identified (12). To test whether the ORF2 was post-translationally modified, including acetylation, GFP-tagged truncated fragments of

genotype

1 and 4 ORF2 (GFP-1-ORF2(112-660) and GFP-4-ORF2(112-660)) were introduced into 293T cells. ORF2 was then immunoprecipitated using GFP antibody and mass spectrometry analysis by acetylation was performed (fig. 1A,1C, 1D). Mass spectrometry results showed that GFP-1-ORF2 and GFP-4-ORF2 were in the same peptide EPTVK⁴¹¹LYTSVEN was acetylated at the K411 position, a sequence well conserved among the 9 ORF2 polypeptide sequences examined (including genotypes 1-4) (FIG. 1B).

Using antibodies against acetylated lysine, it was further verified that acetylation occurred in GFP-1-ORF2 and GFP-4-ORF2, but not on GFP alone (FIG. 1E). To verify that K411 is the acetylation site of ORF2, K411 was mutated to arginine and the GFP-tagged mutant was expressed in 293T cells. Acetylation of the mutant was greatly reduced compared to wild-type ORF2 (fig. 1F), suggesting that K411 is the primary acetylation site of ORF 2.

Example 2: formation of HEV Inclusion bodies depends on K411 acetylation of ORF2

Since lysine acetylation plays an important role in the life cycle of the virus, it was decided to evaluate the function of K411 acetylation of HEV ORF 2. The wild-type 1-ORF2(112-660),4-ORF2(112-660), mutant 1-ORF2 fused to a GFP tag were used^K411R(112-660), or 4-ORF2^K411R(112-660), HeLa cells were transfected. Cells were fixed, immunofluorescent stained with anti-GFP antibody and anti-tubulin antibody, and DNA stained by DAPI. Fluorescence microscopy confirmed the localization of ORF2 in the cells. It was surprisingly found that GFP-1-ORF2^K411R(112-660) and GFP-4-ORF2^K411R(112-660) are mainly uniformly dispersed in the cytoplasm, whereas the wild-type GFP-ORF2(112-660) of genotype 1 and genotype 4 are evidentStrong inclusion formation is shown (fig. 2A, 2B). To examine whether enhanced inclusion body formation was due to N-terminal truncation, full-length ORF2(ORF2(1-660)) was tested, with similar results to the truncated ORF2 described above (FIG. 2C). To rule out the possibility that enhancement of inclusion body formation was caused by GFP tagging, the GFP tagging was replaced with HA or FLAG, and similar results were obtained (fig. 2D). Cells with inclusion bodies (GFP spot count)>3/cell) accounted for 80% and 75% or more of the total number of cells expressing the wild-type GFP-1-ORF2(112-660) and GFP-4-ORF2(112-660) (FIG. 2E). Number of cells having inclusion body in cytoplasm (GFP dot count)>3/cell) appeared comparable for full-length and truncated ORF2 (fig. 2F). However, GFP-1-ORF2^K411RThe mutant had a significantly reduced number of inclusion bodies (fig. 2F).

To verify the above results, the GFP-tagged ORF2 wild-type and K411R mutants (i.e., wild-type 1-ORF2(112-660),4-ORF2(112-660),1-ORF 2)^K411R(112-660), or 4-ORF2^K411R(112-660)), transfecting A549 cells (a cell for HEV culture (41)). These cells exhibited similar results as in HeLa cells (fig. 8).

Taken together, these data indicate that K411 plays an important role in ORF 2-mediated inclusion body formation.

Example 3: dynamic evaluation of the Effect of the mutation K411R on Inclusion body formation

It appears that acetylation of HEV ORF2 is involved in regulating the formation of inclusion bodies (fig. 2). However, the inclusion body formation process of HEV ORF2 is still unknown. To determine the effect of K411 acetylation on this process, live cell imaging of wild-type ORF2 and acetylation deleted ORF2 was performed in HeLa cells and ORF2 inclusion body formation was analyzed. Viable cell imaging began 12 hours after transfection of GFP-1-ORF2(112-660) and continued for as much as 8 hours. At the beginning of imaging, the cells had few detectable inclusions, and a few observable inclusions were formed after about 30-90 minutes (fig. 3A). Thereafter, assembly produced increasingly stronger high volume inclusion bodies over time (fig. 3A). These results demonstrate that acetylation of K411 greatly affects the self-assembly of ORF2 inclusion bodies. To determine whether acetylation is one of the determinants of inclusion body formation, GFP-1-ORF2^K411R(112-660) live cells were imaged 8 hours after transfection, and no examination spot was formed in the cells (FIG. 3B).

Counting in more than 3,000 living cells by a high content cell analyzer ImageXpress Micro XL system, performing inclusion body statistical analysis and SPOT cell calculation mode,>1 point/cell. The results showed that GFP-1-ORF2^K411RSignificantly reduced inclusion body formation compared to wild-type GFP-1-ORF2 (FIG. 9). Furthermore, GFP-4-ORF2 and GFP-4-ORF2 are also disclosed^K411RThe same results were obtained in expressing cells (FIG. 10).

Thus, these data present for the first time the dynamic process of ORF2 formation of inclusion bodies in cells and suggest that acetylation deficiency of K411 would hamper the inclusion body formation process.

Example 4: HDAC6 causes deacetylation of ORF2 and effects Inclusion formation

Acetylation and deacetylation of lysine is dynamically regulated. Histone deacetylase HDAC6 has been previously identified as a molecule that undergoes protein-protein interactions with ORF2 (12). Previous studies have shown that HDAC6 affects protein aggregation and viral infection processes. Therefore, the hypothesis is presented: HDAC6 may play a regulatory role in inclusion body formation by modulating acetylation of ORF 2. Here, both

genotypes

1 and 4 ORF2 were confirmed to physically interact with exogenous and endogenous HDAC6 by immunoprecipitation experiments (fig. 4A and 4B). Furthermore, when HDAC6 was knocked out with siRNA, the amount of HDAC6 decreased, while the amount of acetylated ORF2 significantly increased, indicating that HDAC6 caused deacetylation of ORF2, while inhibition of HDAC6 expression may increase the amount of acetylated ORF2 (fig. 4C). Further, using 2 small molecule inhibitors Trichostatin a (TSA) and Tubastatin a (TBSA) of HDAC6, it was also examined whether blocking HDAC6 activity would affect the acetylation and inclusion body formation of ORF 2. Although TSA is a non-specific HDAC inhibitor, TBSA is highly selective for HDAC6 (42, 43). As a result, it was found that acetylated GFP-1-ORF2 and GFP-4-ORF2 were increased after 8 hours of TBSA treatment compared to DMSO control treatment (FIG. 4D). Treatment with TSA also up-regulated the GFP-1-ORF2(112-660) and GFP-4-ORF2(112-660) protein levels (FIG. 12). Importantly, cells treated with HDAC6inhibitor formed significantly more inclusion bodies and the inclusion bodies were more voluminous compared to DMSO control treatment (fig. 4E, 4G). Notably, TBSA-treated cells showed a more dense distribution of inclusion bodies in the cells (fig. 4E,4G), probably due to the high potency inhibition of HDAC6 by TBSA. GFP-4-ORF2 also showed a similar cellular phenotype after TSA and TBSA treatment (FIGS. 4F, 4H). Cell images of 293T cells treated with DMSO, TBSA or TSA 24 hours after transfection with GFP-1-ORF2 are shown in FIG. 11.

These results above demonstrate that HDAC6 causes deacetylation of ORF2 and negatively regulates ORF2 to form inclusion bodies.

Example 5: acetylation promotes protein stability of HEV ORF2

An important biological function of HDAC6 is to promote protein degradation (13). To investigate whether lysine acetylation affects HEV ORF2 polypeptide levels, siRNA knockdown HDAC6 was used. Indeed, depletion of HDAC6 was found to greatly increase the level of ORF2 (fig. 5A). To reveal the process of ORF2 degradation, protein synthesis was blocked using the protein translation inhibitor cycloheximide (chx) and protein degradation was blocked using the proteasome inhibitor MG 132. The protein level of GFP-1-ORF2 decreased with time in CHX-treated 293T cells, especially after 6 hours CHX treatment (FIG. 13). In contrast, cells treated with both CHX and MG132 showed comparable levels of GFP-1-ORF2 polypeptide, suggesting that the abundance of ORF2 polypeptide is regulated by a proteasome-dependent degradation pathway. These results suggest that ORF2 might be stabilized by acetylation, suggesting that acetylation of ORF2 might increase the ability of ORF2 to resist proteasomal degradation and thereby improve the stability of ORF 2. This is confirmed by the following results: evaluation of GFP-1-ORF2, GFP-4-ORF2, GFP-1-ORF2 in 293T cells^K411RAnd GFP-4-ORF2^K411RThe protein level of (a); the amount of K411 mutant was greatly reduced compared to wild-type ORF2 (fig. 5B, left panel). Furthermore, the difference in protein amounts of wild-type and mutant ORF2 was largely eliminated after 6 hours of treatment of the cells with proteasome inhibitor MG132 (fig. 5B, right panel).

Taken together, these data indicate that: acetylation of K411 can enhance resistance of ORF2 polypeptide to proteasome-dependent degradation, increasing stability of ORF2 polypeptide; furthermore, this stabilization of ORF2 by K411 acetylation could be antagonized by HDAC 6.

To investigate the relationship between protein stability and inclusion body formation of HEV ORF2, cells expressing GFP-1-ORF2 or GFP-4-ORF2 were treated with the HDAC6inhibitor TBSA for 8 hours and monomeric and multimeric ORF2 polypeptides were detected by non-reducing SDS-PAGE (FIG. 5C). Monomeric ORF2 polypeptide did not increase significantly with TBSA treatment compared to DMSO control. In contrast, multimeric ORF2 polypeptides were sharply upregulated with TBSA treatment (fig. 5C). The reason for this increase may be that high acetylation levels favor ORF2 inclusion body formation. The total amount of ORF2 polypeptide after TBSA treatment was also assessed by reducing SDS-PAGE detection (FIG. 5C). These data indicate that high acetylation levels of ORF2 can contribute to protein stability of HEV ORF2 by promoting ORF2 multimerization.

Example 6: inhibition of HDAC6 enhances HEV replication and VLP formation

Since K411 acetylation plays an important role in regulating ORF 2-mediated inclusion body formation and ORF2 polypeptide stability, it was decided to assess whether acetylation affects HEV viral replication. Recently, an efficient cell culture system for HEV genotype 4 has been developed in PLC/PRF/5 cells (44). Thus, the effect of acetylation on HEV virus replication was investigated using this cell culture system by modulating HDAC6 activity using specific HDAC6 inhibitors. To this end, PLC/PRF/5 cells were infected with HEV for 3 weeks, followed by treatment of the cells with HDAC6inhibitor for 3 days. Thereafter, cell culture supernatants were collected and examined for HEV antigen using enzyme-linked immunosorbent assay (ELISA). The results show that the amount of HEV antigen (ORF2) was up-regulated after TBSA treatment compared to DMSO treatment (fig. 6A). This result is consistent with the results obtained using GFP-tagged ORF2 (fig. 5C), suggesting that inhibition of HDAC6 activity may enhance HEV viral replication.

Considering that ORF2 polypeptide expressed from sf9 can also efficiently form VLPs and that the insect cells also have their own acetylation-deacetylation system (45), acetylation of HEV 1-ORF2 VLPs purified from the supernatant of sf9 cells was evaluated by mass spectrometry and it was confirmed that ORF2K411 was acetylated (fig. 6B). Small molecule inhibitors TSA, TBSA and CAY targeting HDAC6 (CAY10603) were added to sf9 cells expressing ORF 2. After 5 days, the cell culture supernatants were tested for ORF2 by ELISA. The results show that all three inhibitors significantly improved ORF2 expression (fig. 6C), suggesting that HDAC 6inhibitors enhanced VLP formation. Indeed, although CAY treatment increased the level of free ORF2, VLPs purified from sf9 cell culture supernatants by ultracentrifugation showed a significant increase after treatment with all three HDAC 6inhibitors compared to DMSO control treatment (fig. 6D, 6E).

In addition, the effect of acetylation on VLP formation was assessed using transmission electron microscopy. GFP-1-ORF2 was found to self-assemble successfully into VLPs (concentrated in the black area) (FIG. 6F). After treatment of cells with TBSA, the cells were incubated with ORF2^K411RThe region of the ORF2 VLPs appears much larger compared to VLPs, further demonstrating that acetylation of K411 can have an important role in ORF2VLP assembly or ORF2VLP stabilization. At GFP-4-ORF2 and GFP-4-ORF2^K411RThese results in expressing cells were similar to those of genotype 1ORF2 (fig. 6G).

Taken together, these data indicate that high acetylation levels of ORF2 promote HEV replication and VLP assembly, while HDAC6 may serve as a host defense mechanism against HEV replication and virion formation.

Discussion:

viral inclusion bodies have been proposed as replication factories or platforms for efficient viral replication (31). In general, inclusion bodies can be formed spontaneously from aggregates of HEV virion or capsid protein ORF2 with cellular proteins. However, it is not known how host proteins regulate capsid protein ORF2 self-assembly and viral inclusion body formation. HEV viruses have a limited genome size, thereby requiring the use of host cell proteins in the life cycle of the virus. The inventors herein report a novel mechanism upon which HEV ORF2 stability maintenance and inclusion body formation are based. The inventors have found that HEV ORF2 is acetylated at the conserved amino acid residue K411 position of genotype 1 and genotype 4. When K411 was mutated to arginine, the ability of ORF2 to form inclusion bodies and the stability of the protein was significantly impaired, suggesting that lysine acetylation is involved in regulating inclusion body formation and ORF2 antigen abundance.

Lysine acetylation plays an important role in the regulation of the viral life cycle. For example, HIV-1 envelope-dependent cell fusion and infection are greatly inhibited when acetylation of alpha-tubulin is inhibited (46, 47). Considering that α -tubulin is acetylated in mammalian cells and microtubules participate in the fusion of human parainfluenza virus type 3 inclusion bodies (31), there is a possibility that the increase of HEV ORF2 inclusion bodies under TSA and TBSA treatment may be partially caused by the inhibition of acetylation of α -tubulin. However, acetylation deleted ORF2^K411RThe mutant lost the ability to aggregate into inclusion bodies, indicating that ORF2 acetylation is essential for this process.

Time-lapse live cell imaging revealed that K411 acetylation of ORF2 had a significant effect on de novo inclusion body self-assembly. Moreover, acetylation of K411 significantly promotes HEV ORF2 resistance to proteasome-dependent degradation pathways by forming multimers, promoting the stability of HEV ORF 2. Thus, the inventors' findings reveal a novel mechanism for modulating HEV ORF2 stability and IB formation by a host lysine acetylation system (fig. 7). Although the acetyltransferase responsible for acetylating the K411 residue of ORF2 has yet to be identified, current findings suggest that HDAC6 appears to antagonize ORF2 acetylation. To the best of the inventors' knowledge, HEV ORF2 is the second viral protein target of HDAC6 identified thus far. Consistent with the deacetylation results of ORF2, inhibition of HDAC6 greatly enhanced ORF2 polypeptide stability, inclusion body formation, HEV replication, and virus-like particle formation. This suggests that HDAC6 may exert a host defense mechanism against HEV infection by antagonizing ORF2 acetylation. Thus, modulation of acetylation of ORF2 may be a novel method for HEV treatment in critically ill patients and chronic HEV infected patients. In addition, the use of HDAC 6inhibitors may improve HEV vaccine production efficacy.

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Claims

1. A method of improving HEV virus production, comprising: culturing HEV infected cells in the presence of an HDAC6inhibitor,

wherein the inhibitor knockdown HDAC6 expression and/or reduces HDAC6 activity in the cell,

wherein the HEV virus is a genotype 1or 4 HEV virus.

2. A method of improving HEV virus-like particle production, comprising: culturing a cell comprising a recombinant expression construct comprising a nucleic acid encoding an ORF2 polypeptide in the presence of an HDAC6inhibitor,

wherein the ORF2 polypeptide comprises the peptide sequence E (P/L) TVK⁴¹¹LYTSVEN, and is selected from the following sequences:

(a) the full-length sequence of HEV ORF2 protein of genotype 1or 4;

(b) a truncated fragment of (a), said fragment consisting of the amino acid sequence aa 112-660.

3. A method of improving HEV ORF2 polypeptide production, comprising: culturing a cell comprising a nucleic acid encoding an HEV ORF2 polypeptide in the presence of an HDAC6inhibitor,

(a) the full-length sequence of HEV ORF2 protein of genotype 1or 4;

4. The method of claim 2 or 3, wherein said ORF2 polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4.

5. The method of any one of claims 1 to 3, wherein the level of expression of HDAC6in the cell is knocked down.

6. The method of claim 5, wherein the knockdown of HDAC6 levels is achieved using a nucleic acid molecule that inhibits the expression of HDAC 6.

7. The method of claim 6, wherein the nucleic acid molecule that inhibits the expression of HDAC6 is an siRNA or shRNA or an expression construct thereof.

8. The method of claim 5, wherein HDAC6 expression is knocked down prior to viral infection.

9. The method of claim 5, wherein HDAC6 expression is knocked down prior to expression of ORF 2.

10. The method of any one of claims 1-3, wherein the cell is an HDAC6 knockdown cell.

11. The method of claim 10, wherein the cell is an HDAC6 constitutive knockdown cell or an HDAC6 conditional knockdown cell.

12. The method of any one of claims 1 to 3, wherein the cell is treated with an HDAC6 chemical inhibitor to reduce HDAC6 activity in the cell.

13. The method of claim 12, wherein the chemical inhibitor of HDAC6 is selected from the group consisting of the following non-selective inhibitors: TSA, Vorinostat, LBH589, ITF2357, PXD-101, or a selective inhibitor selected from HDAC 6: rocilinostat (ACY-1215), ACY-241, Tubacin, Tubastatin A (TBSA), CAY10603, Nexturastat A, HPOB, ACY-738, ACY-775, and ACY-1083, or any combination thereof.

14. The method of claim 12, wherein the cells are treated with an HDAC6inhibitor selected from the group consisting of:

(a) trichostatin A (TSA) at a concentration of 0.2. mu.M to 5. mu.M,

(b) tubastatin A (TBSA) with the concentration of 1-10 μ M,

(c) CAY10603 at a concentration of 0.1. mu.M to 1. mu.M.

15. The method of claim 14, wherein the cells are treated with Trichostatin a (TSA) at a concentration of 1 μ M.

16. The method of claim 14, wherein the cells are treated with Tubastatin a (TBSA) at a concentration of 6 μ Μ.

17. The method of claim 14, wherein the cells are treated with CAY10603 at a concentration of 0.2 μ Μ.

18. The method of any one of claims 1-3, wherein the level of K411 acetylation of HEV ORF2 protein in the cell is increased relative to a cell not treated with the HDAC6 inhibitor.

19. The method of any one of claims 1-3, wherein the stability of ORF2 in the cell and/or the amount of ORF2 in the cell is increased relative to a cell not treated with the HDAC6 inhibitor.

20. The method of claim 19, wherein the amount of ORF2 multimer in the cell is increased relative to a cell not treated with the HDAC6 inhibitor.

21. The method of any one of claims 1-3, wherein HEV virus replication and/or HEV virus production is increased in the cell relative to a cell not treated with the HDAC6 inhibitor.

22. The method of any one of claims 1-3, wherein VLP assembly and/or VLP production in the cell is increased relative to a cell not treated with the HDAC6 inhibitor.

An in vitro use of an HDAC6inhibitor,

(a) for increasing K411 acetylation level of HEV ORF2,

(b) for increasing HEV ORF2 stability and/or ORF2 yield,

(c) for increasing HEV virus replication and/or virus yield, or

(d) For increasing the assembly of HEV VLPs and/or VLP production.

24. The use of claim 23, wherein the HDAC6inhibitor is an HDAC6 inhibitory nucleic acid or an HDAC6 chemical inhibitor.

25. A cell culture system, comprising:

(i) a cell comprising a nucleic acid encoding an ORF2 polypeptide; and (ii) an inhibitor of HDAC6,

(a) the full-length sequence of HEV ORF2 protein of genotype 1or 4;

26. The cell culture system of claim 25, wherein the inhibitor is an HDAC6 inhibitory nucleic acid molecule introduced into the cell.

27. The cell culture system of claim 26, wherein the HDAC6 inhibitory nucleic acid molecule is an siRNA or shRNA or expression construct thereof.

28. The cell culture system of claim 25, wherein the inhibitor is an HDAC6 chemical inhibitor added to a culture of the cells.

29. The cell culture system of claim 28, wherein the HDAC6 chemical inhibitor is an HDAC6 selective inhibitor.

30. The cell culture system of claim 25, wherein the cell is a HEV virus infected cell of genotype 1or 4.

31. The cell culture system of claim 25, wherein said cell is a recombinant cell into which has been introduced an expression construct expressing said ORF2 polypeptide.

32. Use of a cell culture system according to any one of claims 25-31 for the production of a virus, virus-like particle or ORF2 polypeptide.

33. A cell culture comprising an HDAC6 knockdown cell, wherein the cell comprises a nucleic acid encoding HEV ORF2,

wherein the ORF2 polypeptide comprises the peptide sequence E (P/L) TVK⁴¹¹LYTSVEN and is selected from the following sequences:

(a) the full-length sequence of HEV ORF2 protein of genotype 1or 4;

34. The cell culture of claim 33, wherein said cell is an HEV-infected cell or a cell comprising an expression construct expressing an ORF2 polypeptide.

35. The cell culture of claim 33, wherein the cells comprise a nucleic acid molecule that inhibits the expression of HDAC 6.

36. The method according to claim 35, wherein in particular said nucleic acid molecule inhibiting the expression of HDAC6 is an siRNA or shRNA or an expression construct thereof.

37. Use of a cell culture according to any one of claims 33 to 36 for the production of HEV virus, virus-like particle or ORF2 polypeptide.

38. Use of a virus obtained according to the process of claim 1, or a virus-like particle obtained according to the process of claim 2, or an ORF2 polypeptide obtained according to the process of claim 3, for the preparation of an immunogenic composition or a vaccine.