BRIEF REPORT

High-yield production of canine parvovirus virus-like particlesin a baculovirus expression system

Hongli Jin1 • Xiaohong Xia1 • Bing Liu1 • Yu Fu1 • Xianping Chen1 •

Huihui Wang1 • Zhenqiang Xia1

Received: 14 August 2015 / Accepted: 2 December 2015 / Published online: 14 December 2015

� Springer-Verlag Wien 2015

Abstract An optimized VP2 gene from the current

prevalent CPV strain (new CPV-2a) in China was expres-

sed in a baculovirus expression system. It was found that

the VP2 proteins assembled into virus-like particles (VLPs)

with antigenic properties similar to those of natural CPV

and with an especially high hemagglutination (HA) titer

(1:220). Dogs intramuscularly or orally immunized with

VLPs produced antibodies against CPV with [1:80

hemagglutination inhibition (HI) units for at least 3

months. The CPV VLPs could be considered for use as a

vaccine against CPV or as a platform for research on chi-

meric VLP vaccines against other diseases.

Keywords Canine parvovirus � Virus-like particles �Vaccine

Canine parvovirus (CPV) is a non-enveloped, single-

stranded DNA virus that can cause severe myocarditis,

enteritis and lymphopenia in dogs. Several CPV variants

have emerged since it has spread worldwide, including

CPV-2, CPV-2a, CPV-2b, CPV-2c, new CPV-2a, new

CPV-2b, CPV-2c(a) and CPV-2c(b). Vaccination to protect

dogs from CPV infection is highly important. Most

licensed vaccines were modified based on the original virus

type CPV-2 or CPV-2b. Pratelli et al. [22] showed that

pups inoculated with CPV-2 had lower antibody titers to

heterologous virus (CPV-2b) compared homologous virus

(CPV-2). Cavalli et al. [6] evaluated the antigenic rela-

tionships among CPV-2, CPV-2a, CPV-2b and CPV-2c and

indicated that immunization with CPV-2 resulted in lower

antibody titers against heterologous virus than homologous

virus. All of these lines of evidence suggest that traditional

vaccines may not provide optimal protection against

heterologous virus. Vaccine failure may occur in regularly

vaccinated dogs (even adult dogs) due to low levels of

antibody titers against variant viruses [3, 5, 8]. Thus, many

researchers have suggested the necessity of updating CPV

vaccines to contain the current prevalent strains [3, 8, 25].

New CPV-2a and new CPV-2b types are the prevalent

strains in China, and the new CPV-2a type is the pre-

dominant CPV type [26, 28]. Thus, the development of a

new and safe vaccine based on the current prevalent anti-

genic type is urgently needed considering the high rate of

generation of new CPV strains [25].

Virus-like particles (VLPs) can be obtained by the self-

assembly of one or several viral structural proteins pro-

duced in expression systems. The outstanding advantages

of VLPs (or well-made inactivated CPV vaccines) are their

safety and high immunogenicity, and they could open a

new frontier in vaccine development [7]. VP2, the major

capsid protein of CPV, plays an important role in stimu-

lating protective immunity. Previous studies have shown

that CPV-2 VP2 expressed alone is incorporated efficiently

into empty capsids that retain biological properties of

natural CPV [23, 27]. Elia et al. [9] expressed CPV VLPs

in an insect-baculovirus system and developed an enzyme-

linked immunosorbent assay (ELISA) based on rVP2. Feng

et al. [10] expressed CPV VLPs in silkworm pupae and

confirmed that these VLPs elicited both cellular and

humoral immune responses. One of the limitations in the

development of a successful vaccine is the inability to

generate high levels of immunogenic proteins. Therefore,

& Hongli Jin

jin8616771@163.com

& Zhenqiang Xia

ccxn01@hotmail.com

1 Changchun SR Biological Technology Co., LTD,

Changchun 130012, China

123

Arch Virol (2016) 161:705–710

DOI 10.1007/s00705-015-2719-1

in this study, we aimed to generate CPV VLPs based on the

current prevalent CPV type in high yield and test their

applicability as a vaccine.

We chose the VP2 gene of new CPV-2a type as the

template for developing the vaccine. The levels of

expression of functional proteins can be low because of the

non-canonical genetic code used by this virus and expres-

sion-limiting regulatory elements within the coding

sequence. Codon optimization is an effective method for

solving this problem [24, 29]. This method enhances pro-

tein production by correlating preferred codons with the

abundance of cognate tRNAs available within cells [16]

and can disrupt cis-acting negative regulatory sequences

[13]. Moreover, codon optimization can increase the

immunogenicity of target proteins, which is the goal of

DNA vaccine research [14].

In this study, we optimized the codons of CPV VP2 for

Spodoptera frugiperda 9 (Sf9) cells without inducing any

amino acid changes. The optimized VP2 gene sequence

was amplified using two pairs of primers and cloned into

the pFastBac Dual vector (Invitrogen, Carlsbad, CA, USA)

to generate pFD-VP2, which contained two copies of VP2

and was used as a donor plasmid. Escherichia coli

DH10Bac competent cells (Invitrogen, Carlsbad, CA,

USA) were transformed with the donor plasmid pFD-VP2

to generate a recombinant bacmid containing the target

gene. Sf9 cells were seeded in a 6-well plate at 9 9 105

cells/well and were transfected with 8 lg of recombinant

bacmid using ‘‘Lipofectamine’’ 2000 Transfection Reagent

(Invitrogen, Carlsbad, CA, USA) to produce recombinant

baculovirus according to the manufacturer‘s instructions.

After two consecutive passages, virus-infected Sf9 cells

typically displayed phenotypic changes such as an increase

in cell diameter, the appearance of vesicles and detachment

from the plate (Fig. 1Aa). In contrast, uninfected Sf9 cells

did not exhibit any changes in morphology (Fig. 1Ab).

After three consecutive passages, recombinant baculovirus

in the harvested supernatants was titrated by plaque assay

[17]. Approximately 9.2 9 107 plaque-forming units were

obtained per ml of virus (pfu/ml). A bacmid control (a

product of transformation using the pFastBac Dual vector

as the donor plasmid) was used to produce a baculovirus

control.

The expression of VP2 was detected using an indirect

fluorescence assay (IFA). A monoclonal mouse anti-CPV

antibody (HyTest Ltd., Turku, Finland) was used as a

primary antibody, and fluorescein isothiocyanate (FITC)-

conjugated goat anti-mouse IgG (Sigma, St. Louis, MO,

USA) was used as a secondary antibody. As shown in

Fig. 1B, specific green fluorescence was observed in the

positive wells (Fig. 1Ba) but not in the uninfected wells

(Fig. 1Bb). After negative staining with uranyl acetate, the

samples were examined by electron microscopy. VLPs

were observed (Fig. 1C), and the morphology and size

Fig. 1 Generation of recombinant baculovirus and identification of

VP2 proteins expressed in Sf9 cells. (A) Infection with recombinant

baculovirus changes the morphology of Sf9 cells. (a) Uninfected cells

and (b) cells infected with recombinant baculovirus for 72 h were

observed under a light microscope (magnification, 1009). (B) Indirect

fluorescence assay (IFA) of VP2 protein expressed in Sf9 cells.

(a) Cells infected with the recombinant baculovirus for 48 h and

(b) uninfected cells were identified by IFA and observed under a

fluorescence microscope (magnification at 1009). (C) Electron

micrograph of CPV VLPs. The bar represents 200 nm. (D) Western

blot analysis of VP2 expressed in Sf9 cells. Lane 1, cells infected with

the recombinant baculovirus expressing VP2 of CPV; lane 2, pre-

stained protein markers; lane 3, cells infected with the baculovirus

control. (E) SDS-PAGE analysis of purified CPV VLPs. Lane 1,

purified CPV VLPs; lane 2, unpurified products; lane 3, cells infected

with the baculovirus control; lane 4, pre-stained protein markers

706 H. Jin et al.

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(approximately 26 nm) of the VLPs were similar to those

of natural parvovirus particles. The immunoreactivity of

the expressed CPV VP2s was examined using a western

blot assay (Fig. 1D). A polyclonal dog anti-CPV-VP2

antibody (prepared in our laboratory) was used as a pri-

mary antibody, and horseradish peroxidase (HRP)-conju-

gated goat anti-dog IgG (Sigma, St. Louis, MO, USA) was

used as a secondary antibody. 3,30-Diaminobenzidine

(DAB; Sigma, St. Louis, MO, USA) was added as a

chromogen for staining. As shown in Fig. 1D, a single

band with a molecular weight of approximately 67 kDa

was present in lane 1, and no specific band was observed in

lane 3, which was the control lane. The results indicate that

the recombinant proteins retained VP2-specific

immunoreactivity. Infected Sf9 cells cultured in suspension

in Erlenmeyer flasks kept on orbital shakers at 120 rpm and

27 �C were harvested at 4 days postinfection to determine

the HA activity of the expressed CPV VLPs as described

previously [10]. The results showed that the HA activity of

CPV VLPs expressed in Sf9 cells was as high as 1:220

(Fig. 2A), and the yield was significantly higher than that

obtained using cultivated mammalian cells (Fig. 2B) or

that previously reported for baculovirus expression systems

[9, 10]. The uninfected Sf9 cells were negative for HA

(Fig. 2). Thus, it is possible to harvest a large amount of

CPV VLPs with a high HA titer due to the high expression

and suspension characteristics of Sf9 cells. To confirm the

yield of CPV VLPs, we harvested infected Sf9 cells cul-

tured in suspension and purified the VLPs as described

previously [4]. The purified protein was characterized by

SDS-PAGE (Fig. 1E) and scanned using a thin layer

chromatogram scanner to estimate the purity of the protein,

which was found to be above 90 %. The concentration of

purified protein was measured using a BCA protein assay

kit (Thermo, Waltham, MA, USA). The results showed that

90.3 mg of purified VLPs could be obtained from 1L

suspension products.

To investigate the immunogenicity of the CPV VLPs, a

vaccine was prepared. Sf9 cells were infected with

recombinant baculovirus at a multiplicity of infection

(MOI) of 1. Cells were cultured in suspension, harvested at

4 days postinfection, and lysed at 4 �C in 25 mM bicar-

bonate solution for 30 min. Cell debris was removed by

low-speed centrifugation.

The cost of vaccines should be considered when they are

used in veterinary medicine, and a cost-effective approach

should be carried out instead of using expensive equip-

ment. Furthermore, baculoviruses are non-infectious and

safe for use in mammals, and they can be used as adjuvants

to induce innate immunity [1, 15, 18]. Therefore, after

discarding cell debris, we diluted the antigens, which

included CPV VLPs, instead of performing further purifi-

cation steps (such as sucrose density gradient centrifuga-

tion). A dilution could be used to prepare the vaccine

considering the high yield (HA = 1:220) of CPV VLPs

obtained in this study. Samples with suitable HA titers

(1:210 and 1:212) were prepared by dilution with PBS, and

then the diluted samples were divided into two groups. The

Fig. 2 Hemagglutination (HA) activity of CPV VLPs. HA titers were detected using pig erythrocytes. (A) Recombinant baculovirus cultivated

in Sf9 cells; (B) CPV strain cultivated in F81 cells; (C) uninfected Sf9 cells

Canine parvovirus virus-like particles 707

123

samples in one group were mixed with Al(OH)3 adjuvant

(final concentration of Al(OH)3, 4 %) and used as intra-

muscularly administered (IM) vaccines. Those in the other

group were used as orally administered (OR) vaccines.

Thirty dogs were divided into the following five groups:

a low-dose IM group, a high-dose IM group, an IM control

group, an OR group and an OR control group. The low-

dose IM and high-dose IM groups were immunized with

the IM vaccine prepared as described above with HA units

of 1:210 and 1:212, respectively. Each dog received 1 ml of

vaccine and was boosted with the same vaccine two weeks

later. The IM control group received the baculovirus con-

trol. The OR group was immunized with 2 ml of OR

vaccine with 1:212 HA units and received an immunization

boost with the same dose two weeks later. The OR control

group received the baculovirus control in the same manner

as the OR group. To determine whether the vaccines were

safe for the animals, parameters such as feces production,

behavior, and food and water consumption were observed

daily to identify any abnormalities. Serum samples were

collected at 0, 1, 2, 3, 4, 8 and 12 weeks after immuniza-

tion to determine antibody titers against CPV using the

hemagglutination inhibition (HI) test as described previ-

ously [10].

No adverse reactions were observed in the experimental

groups after immunization of the dogs with CPV VLPs.

The results show that the antigens that we constructed are

safe for dogs. Both the IM and OR groups that were

administered CPV VLPs developed antibodies, and the

antibody levels peaked at 1-2 weeks after the second

immunization. The total antibody titer of the dogs in the

OR group was lower than that of the dogs in the IM groups.

In addition, the antibody titer (HI titer) was high ([1:80)

and was sustained for at least 3 months (Fig. 3). The

observed strong immune responses may have occurred

because the CPV VLPs are of a size that allows them to be

taken up by dendritic cells (DCs). There was no significant

difference between the HI titers of the high-dose IM group

and those of the low-dose IM group (Fig. 3A). Although

the OR group was not injected with an adjuvant, the group

received a larger dose of vaccine compared with the IM

group. Dogs from the OR group produced a lower HI titer

after the first immunization compared with those of the IM

group. However, the antibody titer of the OR group greatly

increased after administration of a booster immunization

2 weeks after the first dose (Fig. 3B). Previous reports have

shown similar immune responses to vaccines given by the

oral route. Gil et al. [11] expressed a 21-mer peptide (2L21,

derived from CPV VP2 protein) in transgenic plants fusion

with b-glucuronidase gene. After oral immunization with

transgenic plant extracts, the mice produced 2L21 and

VP2-specific antibodies. Gil et al [12] also fused the

21-mer peptide to a 41-amino-acid-long tetramerization

domain (TD) from the transcriptional factor p53. The

chimerical DNA construction 2L21-TD was cloned in a

binary plant transformation vector and used to transform

Arabidopsis thaliana plants. Mice immunized by the oral

route with crude protein produced CPV-specific antibodies.

Feng et al. [10] expressed CPV VLPs in silkworm pupae

and showed that a systemic immune response could be

elicited in dogs that orally received 2 ml of raw homo-

genates. These results indicate that CPV VLPs remain

immunogenic when they are presented to the gut mucosal

immune system. Gastric acid and enzymatic digestion,

which usually interfere with the functioning of OR vacci-

nes, may not be issues with this vaccine, likely because

Fig. 3 Immunogenicity of CPV VLPs in dogs as determined by

hemagglutination inhibition (HI) antibody titers. (A) Dogs were

immunized by intramuscular (IM) injection. The low-dose IM and

high-dose IM groups were immunized with CPV VLPs mixed with

Al(OH)3 adjuvant with 1:210 and 1:212 HA units, respectively. Two

weeks later, dogs received an immunization boost. The IM control

group was immunized with a baculovirus control. (B) The orally

administered (OR) group was immunized orally with CPV VLPs with

1:212 HA units and received an immunization boost two weeks later.

The OR control group was immunized with a baculovirus control.

After immunization, serum samples from all groups were analyzed by

HI

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CPV VLPs inherit the stability of CPV [10–12]. However,

the mechanism by which immunity is induced is not well

understood. The antibody titers against CPV in the IM

control group and OR control group were low and could be

regarded as negative. Previous in vivo studies have shown

that dogs are protected from infection when the HI anti-

body titer in blood reaches 1:80 [2, 19–21]. Considering

the information that was already available from previous

studies, as well as and ethical issues, we decided not to

perform a challenge test with lethal CPV.

During vaccine preparation, the level of protein impu-

rities was reduced by dilution of the sample, but the CPV

VLPs level was still sufficient to activate a strong immune

response. Therefore, the cost of production of vaccines

using these suspension culture and dilution methods will be

low. The antigen level in the vaccine was sufficient to

produce an immune response because of the high level of

CPV VLPs, in contrast to traditional vaccines in which

insufficient amounts of antigen can result in vaccination

failure. Moreover, the immunization dose was determined

based on the HA unit before immunization, and this

method is easy and has been shown to be appropriate for

judging the amount of immunogen in the preparation [10].

Thus, the methods described here are easy, cost-effective,

and convenient to perform.

In summary, in this study, CPV VLPs were generated

with a particularly high HA titer using a baculovirus

expression system. After IM or OR immunization, the

VLPs triggered a strong immune response. The antigenic

properties of CPV VLPs both in vitro and in vivo will allow

the development of new types of CPV vaccines and CPV

VLP-based chimeric vaccines for other animal diseases.

Moreover, the application of this method is easy and cost-

effective.

Acknowledgments This work was supported by Innovation Fund

for Technology Based Firms of Jilin Province (SC201407103).

Compliance with ethical standards

Conflict of interest The authors do not have any conflict of interest.

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