U.S. patent application number 13/600543 was filed with the patent office on 2013-09-26 for method for preparing virus-like particle and recombinant baculovirus used therein.
This patent application is currently assigned to NATIONAL TSING HUA UNIVERSITY. The applicant listed for this patent is YU-CHEN HU, SHIH-YEH LIN. Invention is credited to YU-CHEN HU, SHIH-YEH LIN.
Application Number | 20130252311 13/600543 |
Document ID | / |
Family ID | 49189440 |
Filed Date | 2013-09-26 |
United States Patent
Application |
20130252311 |
Kind Code |
A1 |
HU; YU-CHEN ; et
al. |
September 26, 2013 |
METHOD FOR PREPARING VIRUS-LIKE PARTICLE AND RECOMBINANT
BACULOVIRUS USED THEREIN
Abstract
A recombinant baculovirus is provided for preparing picornavirus
virus-like particles (VLP), wherein Chitinase A (ChiA) and
Cathepsin V (v-cath) genes of the recombinant baculovirus are
functionally disrupted and the recombinant baculovirus includes a
picornavirus capsid protein gene under control of a strong
promoter, and includes a protease gene configured for encoding a
protease for hydrolyzing the capsid protein under control of a weak
promoter. The recombinant baculovirus of the present invention may
adopt High Five or Sf-9 cells for manufacturing enterovirus
virus-like particles with improved stability and higher yields in
comparison with the conventional arts. A method for preparing virus
like particles is also herein provided.
Inventors: |
HU; YU-CHEN; (HSINCHU,
TW) ; LIN; SHIH-YEH; (HSINCHU, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HU; YU-CHEN
LIN; SHIH-YEH |
HSINCHU
HSINCHU |
|
TW
TW |
|
|
Assignee: |
NATIONAL TSING HUA
UNIVERSITY
HSINCHU
TW
|
Family ID: |
49189440 |
Appl. No.: |
13/600543 |
Filed: |
August 31, 2012 |
Current U.S.
Class: |
435/235.1 |
Current CPC
Class: |
C12N 2770/32062
20130101; A61K 39/00 20130101; A61P 31/14 20180101; C12N 7/00
20130101; C12N 2770/32023 20130101; C12N 2770/32323 20130101; C12N
2710/14143 20130101; C12N 2770/32351 20130101; A61K 2039/5258
20130101; C12N 9/506 20130101 |
Class at
Publication: |
435/235.1 |
International
Class: |
C12N 7/01 20060101
C12N007/01 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 20, 2012 |
TW |
101109435 |
Claims
1. A recombinant baculovirus comprising: a first nucleotide
sequence, for being translated to a capsid protein of a
picornavirus; a first promoter, located in the upstream region of
the first nucleotide sequence; a second nucleotide sequence, for
being translated to a protease of the picornavirus; and a second
promoter, located in the upstream region of the second nucleotide
sequence, wherein the first promoter is a strong promoter and the
second promoter is a weak promoter in the context of recombinant
baculovirus infection to an insect cell and ChiA and v-cath genes
of the recombinant baculovirus are functionally disrupted.
2. The recombinant baculovirus according to claim 1, wherein the
first promoter comprises a baculovirus polyhedrin promoter or a
baculovirus P10 promoter.
3. The recombinant baculovirus according to claim 1, wherein the
second promoter comprises a baculovirus immediate early promoter, a
cytomegalovirus immediate early gene (CMV-1B) promoter or a
Drosophila hsp70 promoter.
4. The recombinant baculovirus according to claim 1, wherein the
second promoter comprises a fragment of the strong promoter.
5. The recombinant baculovirus according to claim 1, wherein the
insect cell comprises a Sf-9 cell strain or a High Five.TM. cell
strain.
6. The recombinant baculovirus according to claim 1, wherein the
picornavirus comprises an enterovirus.
7. The recombinant baculovirus according to claim 6, wherein the
enterovirus comprises enteroviruses 68-71, Coxsackie A viruses or
Coxsackie B viruses.
8. The recombinant baculovirus according to claim 1, wherein the
recombinant baculovirus is used for preparing a vaccine.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field Of The Invention
[0002] The present invention relates to a method for preparing
picornavirus virus-like particles (VLP) and recombinant
baculoviruses used therein, and more particularly to a method for
preparing enterovirus virus-like particles and recombinant
baculoviruses used therein.
[0003] 2. Description Of The Prior Art
[0004] Enteroviruses belong to the Picornaviridae family and are
positive-single stranded RNA viruses. Infection often happens via
respiratory droplet or stool of an infected person, which can
infect multiple systems and organs of a human body and even can
cause organ failure. By serum neutralization test, enteroviruses
can be classified into Poliovirus (PV), Coxsackie A virus (CAV),
Coxsackie B virus (CBV), Enteric Cytopathogenic Human Orphan virus
(i.e. Echoviruses), enteroviruses 68-71 and so on.
[0005] Being one of the more than 100 enteroviruses serotypes,
enterovirus 71 (EV71) is the major etiological agent responsible
for hand-foot-and-mouth disease (HFMD) in young children and
infants. Children under 5 years of age are particularly susceptible
to severe forms of EV71-associated neurological complications such
as aseptic meningitis, brain stem encephalitis and even death.
Since the 1998 outbreak in Taiwan that caused 405 severe cases and
78 deaths (Ho el al., 1999), in recent years the Asia-Pacific
region (e.g. Taiwan, China, Malaysia, Japan and Vietnam) has
experienced more frequent BV71-associated HFMD epidemics with high
incidence of neurotropic complications and fatality rates (Tee et
el., 2010). In 2008, the HFMD outbreak in Taiwan resulted in 373
severe cases and 14 deaths, all due to EV71 infection. The 2008
outbreak in China also led to 489097 reported cases that included
1125 severe cases, and claimed 126 lives. The increasing frequency
of EV71 epidemics and high fatality rates underscore the urgent
need to develop the vaccines.
[0006] VP1 is the major antigen and is highly variable among
enteroviruses. Based on the sequences of VP1 gene, EV71 is divided
into genogroups A, B and C and subgenogroups within genogroups B
and C (B1-B5 and C1-C5).
[0007] Traditional viral vaccines include inactivated vaccines and
attenuated vaccines, both containing the viral genetic materials
and hence posing potential risks. Virus-like particles (VLPs) are
empty particles consisting of viral structural proteins but devoid
of viral nucleic acids, hence they are non-infectious. VLPs can
generally induce broad and strong immune responses thanks to the
preservation of many essential epitopes. Therefore VLPs have
captured increasing attention as potential vaccine candidates.
[0008] Briefly, for the purpose of preventing enteroviruses
infection, VLP is a promising vaccine candidate. Enterovirus
possesses a positive single-stranded RNA genome that consists of a
single open reading frame (ORF). The ORF expresses a large
polyprotein that can be cleaved into P1, P2 and P3 regions (Brown
and Pallansch, 1995), P1 region encodes the four structural,
proteins VP1, VP2, VP3 and VP4, while P2 and P3 regions encode
other nonstructural proteins (e.g. 2A and 3CD) responsible for
virus replication and virulence (McMinn, 2002). Based on a model
derived from poliovirus, protease 2A autocatalytically cleaves P1
at its N-terminus and liberates P1 from the nascent polyprotein
(Toyoda el al., 1986), while protease 3CD cleaves P1 precursor into
VP1, VP3 and VP0 in trans. These three structural proteins
spontaneously assemble into icosahedral procapsid in an ordered
manner and proceeds through a series of intermediates, followed by
the encapsidation of the RNA genome into the provision. The final
encapsidation step involves the cleavage of VP0 into VP2 and VP4,
therefore the final mature virion consists of 60 copies each of VP1
and VP3, 58-59 copies of VP2 and VP4 and 2-1 copies of VP0.
[0009] Based on the knowledge, the baculovirus expression system
has been previously used to co-express the P1 and 3CD proteins of
EV71 in insect cells (Ho et at., 2003) and the formation of EV71
VLP in the infected insect cells was demonstrated. The recombinant
baculovirus, Bac-P1-3CD, harboring the P1 gene under the polyhedrin
promoter and the 3CD gene under the p10 promoter, is constructed
using the traditional Bac-to-Bac system. Immunization of mice with
the BV71 VLP triggered potent humoral and cellular immune responses
and protected mice against lethal virus infection (Chung et al.,
2008). However, the VLP yield was too low (.apprxeq.10-20
.mu.g/ml), making it less attractive for commercial production.
SUMMARY OF THE INVENTION
[0010] The present invention is directed to a method for preparing
picornavirus virus-like particles and recombinant baculoviruses
used therein to improve the stability and yield of virus-like
particles.
[0011] According to an embodiment of the present invention, a
recombinant baculovirus comprises a first nucleotide sequence, a
first promoter, a second nucleotide sequence and a second promoter.
The first nucleotide sequence is used for being translated to a
capsid protein of a picornavirus. The first promoter is located in
the upstream region of the first nucleotide sequence, wherein the
first promoter is a strong promoter when it is in the context of
baculovirus/insect cell system. The second nucleotide sequence is
used for being translated to a protease of the picornavirus,
wherein the protease is used to hydrolyze the capsid protein. The
second promoter is located in the upstream region of the second
nucleotide sequence and is a weak promoter when in the context of
baculovirus/insect cell system, wherein Chitinase A (ChiA) and
Cathepsin V (v-cath) genes of the recombinant baculovirus are
functionally disrupted.
[0012] The objective, technologies, features and advantages of the
present invention will become more apparent from the following
descriptions in conjunction with the accompanying drawings, wherein
certain embodiments of the present invention are set forth by way
of illustration and examples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The foregoing aspects and many of the accompanying
advantages of this invention will become more readily appreciated
as the same becomes better understood by reference to the following
detailed description, when taken in conjunction with the
accompanying drawings, wherein;
[0014] FIG. 1A is a schematic diagram illustrating recombinant
baculoviruses according to prior arts and an embodiment of the
present invention; and
[0015] FIG. 1B to 1E is a diagram showing experimental results of
the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0016] The present invention is directed to a method for preparing
picornavirus virus-like particles and recombinant baculoviruses
used therein to improve stability and yield of virus-like
particles.
[0017] According to an embodiment of the present invention, a
recombinant baculovirus comprises a first nucleotide sequence, a
first promoter, a second nucleotide sequence and a second promoter.
The first nucleotide sequence is used for being translated to a
capsid protein of a picornavirus. The first promoter is located in
the upstream region of the first nucleotide sequence, wherein the
first promoter is a strong promoter in the context of
baculovirus/insect cell system. The second nucleotide sequence is
used for being translated to a protease of the picornavirus,
wherein the protease is used to hydrolyze the capsid protein. The
second promoter is located in the upstream region of the second
nucleotide sequence and is a weak promoter in the context of
baculovirus/insect cell system, wherein ChiA and v-cath genes of
the recombinant baculovirus are functionally disrupted.
[0018] Glossary:
[0019] Picornavirus: Picornaviruses are RNA viruses and mainly
infect human beings and other animals. Picornaviruses comprise
human rhinovirus, human poliovirus, human coxsackie virus, human
echoviruses, human enterovirus, bovine enterovirus,
encephalomyocarditis virus, encephalitis virus, foot-and-mouth
disease virus, hepatitis A virus and so on.
[0020] Enterovirus: Conventionally, enteroviruses can be classified
into Polioviruses (PV), Coxsackie A viruses (CAV), Coxsackie B
viruses (CBV), Enteric cytopathogenic human orphan viruses (i.e.
Echoviruses, ECHO), enteroviruses 68-71 and so on by serum
neutralisation test. Recently with the development of DNA
sequencing, enteroviruses are alternatively classified into four
types A-D and there are more than one hundred of serotypes so far;
Enterovirus 71 (EV71) is one type thereof.
[0021] An enterovirus genome is approximately 7500 nt in length
including an open, reading frame (ORF, approx. 6600 nt) and two
sides of the enterovirus genome are 5' and 3' untranslated regions
(UTR). The ORF can be separated into three regions: P1, P2 and P3
and can be translated to single polyproteins. P1 comprises capsid
proteins: VP4, VP2, VP3 and VP1, P2 and P3 comprise non-structural
proteins such as protease 2A, 3C and 3CD and polymerase 3D.sup.pol,
which contribute to virus duplication and generate virus
toxicity.
[0022] Strong Promoters and Weak Promoters: People skilled should
know appropriate gene expression sequence acting on insect cells
(Lo, W.-H., Hu, Y.-C. 2009. Regulation of baculovirus-mediated gene
expression, in "Regulation of Viral Gene Expression" (Eli B. Galos,
Eds), Nova Science Publishers, Hauppauge. (ISBN:
978-1-60741-224-3)). In some embodiments, the gene expression
sequence comprises constitutive promoters. The strong promoters
used in insect cells include, but are not limited to, baculovirus
p10 promoters, polyhedrin (polh) promoters, p6.9 promoters and caps
id protein promoters. The weak promoters used in insect, cells
include ie1, ie2, ie0, et1, 39K (aka pp31) and gp64 promoters of
baculoviruses.
[0023] Other non-baculovirus weak, promoters in insect cells
include, but are not limited to, Bombyx mori cytoplasmic actin gene
promoters, Drosophila hsp70 promoters, cytoplasmic actin gene
promoters, .alpha.-1 tubulin promoters, ubiquitin gene promoters,
and cytomegalovirus immediate early (CMV-1E) gene promoters.
[0024] Besides, weak promoters can be obtained by attenuating
strong promoters, for example, using a truncated segment of strong
promoter to attenuate the gene expression ability of strong
promoters.
[0025] ChiA and v-cath: ChiA and v-cath are two adjacent genes in
the Autographa californica multicapsid nuclear polyhedrosis virus
(AcMNPV) gene sequence. ChiA protein is an enzyme expressed in the
late stage of virus replication, with high activity of extracelluar
chitinase and intracellular chitinase; v-cath protein is a protease
which is in the form of papain and similar to Cathepsin L. ChiA is
retained in endoplasmic reticulum (ER) with large quantity after
expression with the assistance of an ER positioning sequence KDEL
of terminal C of ChiA sequence. ChiA assists the maturation of
precursor protein of v-cath (pro V-cath), i.e. converting proV-cath
into active V-cath to be released after the death of infected
cells. Since pro V-cath must be folded to become mature enzyme with
the assistance of ChiA, it is proved that pro V-cath and ChiA have
mutual interaction and are accumulated in ER simultaneously.
Accumulation of ChiA and pro V-cath in ER may block the secretory
pathway of secretory proteins.
[0026] Additionally, ChiA and pro V-cath show chitinase and
cysteine protease activity and are released after the death of
infected cells. Therefore, these two enzymes are reported to be
related to liquefaction of insect bodies after insects are infected
with baculovirus and die. The liquefying process of larvae can be
mitigated when the baculovirus is defective in ChiA and v-cath
genes. Thus, for baculovirus/Bombyx mori expression system, there
are many studies using Bombyx mori nucleopolyhedrovirus (BmNPV)
recombinant baculovirus, with chiA and v-cath genes removed for
generating recombinant proteins, thereby raising the yield of
secretory recombinant proteins and decreasing tissue contamination
after liquefaction of larvae bodies so as to facilitate follow-up
protein purification (Lee et al, Biotechnol Lett 28(9);645-50, 2006
; Li et al. Mol Biol Rep 37(8): 3721-8, 2010; Li et al. Appl
Biochem Biotechnol 165(2):728-36, 2011 ; Li et al. Mol Biol Rep
38(6):3897-902, 2011). Likewise, on the basis of baculovirus/insect
cell expression system, multiple studies have used engineered
AcMNPV defective in chiA and v-cath genes to enhance the yield of
secretory proteins with mitigated protein degradation (Kaba et al.
J Virol Methods 122(1):113-8, 2004; Metz et al. Virol J 8:353,
2011: Possee et al. Biochem Soc Trans 27(6):928-32, 1999). There
are also already commercially available recombinant baculovirus
vector systems on the market, such as flashBACGOLD.TM. (Oxford
Expression Technologies Ltd, Oxford, UK) and BacVector-3000
(Novagen, N.J.).
[0027] Functional Disruption: There is at least one mutation or
structural change in genes to make the genes functionally
disrupted, which are substantially unable to generate functional
gene products, e.g. chiA or v-cath proteins. In addition, deleting
or interrupting necessary transcription unit, polyadenylation
signal or splice-site sequence also can generate functional
disruption genes. Furthermore, other methods (e.g. antisense
nucleotide inhibition of gene expression) can be adopted to achieve
functional disruption.
[0028] Using flashBACGOLD.TM. System To Express Virus-Like
Particles
[0029] Referring to FIG. 1A, which is a schematic diagram
illustrating recombinant baculoviruses according to prior arts and
an embodiment of the present invention. Bac-P1-3CD is the
baculovirus generated previously based on Bac-to-Bac system
(Invitrogen, Carlsbad, Calif.) by Chung et al (Chung et al., 2008;
Hu et al., 2003), wherein P1 is driven by polyhedrin promoter while
3CD is driven by p10 promoter and the v-cath and chiA genes are
functional. By adopting the commercial flashBACGOLD.TM. system,
BacF-P1-3CD is generated in which the capsid protein gene P1 of
subtype C2 of enterovirus 71 is driven by the polyhedrin promoter
and the 3CD protease expression is driven by the P10 promoter.
Under the same infection condition (cell density: 2.times.10.sup.6
cells/ml, virus dosage: 0.1 MOI), High Five.TM. cells are infected
by Bac-P1-3CD or BacF-P1-3CD virus and cultured in the shake flask
for protein expression. The supernatant is then collected at 2-6
days after infection. Expression and yield of proteins were
determined by Western blot analysis and Enzyme-Linked immunosorbent
assay (ELISA).
[0030] Referring to FIG. 1B, according to the Western blot analysis
results, BacF-P1-3CD virus constructed using the flashBACGOLD.TM.
system does not enhance the expression of VP1 (which results from
the cleavage of P1 by 3CD and is an. indicator of VLP yield),
instead the VP1 expression by BacF-P1-3CD is poorer than that by
Bac-P1-3CD virus constructed using the Bac-to-Bac.RTM. system. The
ELISA analyses show that the extracellular VLP concentration
conferred by Bac-P1-3CD virus is approximately 18 mg/L and the
extracellular VLP concentration conferred by BacF-P1-3CD virus is
approximately 5 mg/L. Therefore, the recombinant baculovirus
defective in ChiA and v-cath genes constructed using the
flashBACGOLD.TM. system fails to enhance the EV71 VLP yield when
compared with the recombinant baculovirus with functional ChiA and
v-cath genes (Bac-P1-3CD). That is, merely using the
flashBACGOLD.TM. system cannot, necessarily enhance the VLP yield
conferred by the recombinant baculovirus.
[0031] Using Weak Promoters To Drive 3CD Protease Expression
[0032] Chung et al. (Chung et al. Vaccine 28(43):6951-7, 2010)
constructed recombinant baculovirus vectors Bac-P1-C3CD (FIG. 1A)
and Bac-P1-13CD (not illustrated) using weak promoters such as CMV
promoter and baculovirus IE1 promoter to drive the expression of
3CD protease. Such design, was based on the hypothesis that reduced
3CD expression can enhance the P1 expression level, because 3CD
expression driven by a weak promoter would, be less capable of
competing with P1 expression driven by a strong promoter for
cellular resources and thus boost the EV71 VLP yield (Chung et al.
Vaccine 28(43):6951-7, 2010). Indeed, Bac-P1-C3CD (using weak
CMV-1E promoter to drive 3CD expression) has achieved higher yield
than Bac-P1-3CD (using strong p10 promoter to drive 3CD
expression). However, as observed by the inventors, Bac-P1-C3CD
infection resulted in abundant degradation products starting from 4
days post-infection as shown in FIG. 1C. The degradation would not
be able to enhance the VLP yield and instead cause difficulties for
VLP purification. Although we have attempted to increase the yield
by means such as modulating the relative expression levels of P1
and 3CD proteins (data not shown), the yield was only moderately
increased and the VLP degradation was severe. Therefore, there is
still room to improve the VLP yield.
[0033] Using IE1 And CMV Promoters To Drive 3CD Protease In The
FlashBACGOLD.TM. System
[0034] Referring to FIG. 1A and 1B, recombinant baculoviruses
BacF-P1-I3CD and BacF-P1-C3CD are constructed using the
flashBACGOLD.TM. system, which use IE1 and CMV promoters to drive
3CD protease, respectively. Under the same condition as above, VLP
yield was determined after insect cells were infected with these
two strains of recombinant viruses. Western blot analysis (FIG. 1B)
illustrates dramatic increase in the VP1 yield starting from day 4
after the cells are infected with BacF-P1-I3CD and BacF-P1-C3CD
(when compared with Bac-P1-3CD and BacF-P1-3CD). Referring to FIG.
1C, Bac-P1-C3CD virus causes obvious VP1 protein degradation at 5
and 6 days post-infection (dpi). In contrast, the VP1 produced by
BacF-P1-C3CD is more stable with fewer degradation products,
thereby significantly elevating the VP1 yield.
[0035] Referring to FIG. 1D, the actual VLP yield in the
supernatant is determined by ELISA. At the fourth day, the average
VLP yield by Bac-P1-3CD is approximately 18 mg/L and the yield by
BacF-P1-3CD viruses is 5 mg/L. At the sixth day, the yield
conferred by BacF-P1-BCD can reach 100 mg/L. Strikingly, the yield
conferred by BacF-P1-C3CD amounts to 170 mg/L, which is almost 10
times and 34 times the yield conferred by Bac-P1-3CP and
BacF-P1-3CD, respectively.
[0036] From the aforementioned data, it could be observed from
BacF-P1-3CD that merely removing ChiA and v-cath genes from the
baculovirus backbone, as in the case of using the flashBACGOLD.TM.
system, cannot enhance the VLP yield. In the present invention,
Bac-P1-C3CD infection was found to generate a large amount of
degradation products. The data, demonstrate that using the
flashBACGOLD.TM. system or driving 3CD by a weak promoter along
cannot enhance the VLP yield. In contrast, BacF-P1-C3CD, which
combines the use of weak promoter to drive 3CD expression and
disrupted ChiA/v-cath genes, can solve the degradation problem and
achieve increased yield.
[0037] Comparison Of VLP Yield In High Five.TM. And Sf-9 Cells
[0038] High Five.TM. and Sf-9 are two insect cell lines commonly
used for protein production. According to prior research of Chung
et al. (Chung et al. Vaccine 28(43):6951-7, 2010), the highest VP1
protein yield (45 mg/L) was achieved by infecting Sf-9 cells with
Bac-P1-C3CD, and was obtained on the fourth day after Sf-9 cells
were infected with Bac-P1-C3CD. When High Five.TM. cells were
infected with Bac-P1-C3CD, the highest yield of VP1 protein
(.about.30 mg/L) was obtained on the second day. Therefore, in
Chung's system, Bac-P1-C3CD infection in Sf-9 cells would generate
higher VP1 protein yield than Bac-P1-C3CD infection in High
Five.TM. cells.
[0039] As shown in FIG. 1E, BacF-P1-C3CD infection of Sf-9 cells
results in the highest extracellular yield of 100 mg/L at 6 dpi and
the highest intracellular VLP yield of 70 mg/L at 4 dpi. In
contrast, BacF-P1-C3CD infection of High Five.TM. cells results in
significantly higher extracellular VLP yield of 170 mg/L (at 6 dpi)
and intracellular VLP yield of 160 mg/L at 3 dpi. Therefore, the
method for preparing VLP in the present invention is applicable for
both High Five.TM. and Sf-9 cells and the yields thereof are much
higher than those of Chung et al. In addition. High Five.TM. can
result in significantly higher yield than Sf-9, after infection
with the recombinant baculovirus of the present invention, thus
High Five.TM. is preferable for VLP production in the expression
system of the present invention.
[0040] In conclusion, the method and recombinant baculovirus used
for preparing VLP of the present invention, is achieved by using
BacF-P1-I3CD and BacF-P1-C3CD recombinant baculovirus to infect
High Five.TM. cells or Sf-9 cells to generate enterovirus VLP,
wherein the VLP generated is more stable with higher yield than
both prior arts and methods that merely uses flashBACGOLD.TM., i.e.
BacF-P1-3CD.
[0041] While the invention is susceptible to various modifications
and alternative forms, a specific example thereof has been shown in
the drawings and is herein described in detail. It should be
understood, however, that the invention is not to be limited to the
particular form disclosed, but on the contrary, the invention is to
cover all modifications, equivalents, and alternatives falling
within the spirit and scope of the appended claims.
* * * * *