IMMUNOLOGY
ORIGINAL ARTICLE
Immunogenicity of recombinant human bocavirus-1,2 VP2 gene virus-like particles in mice Zhong-Hua Deng,1,2 Ye-Xia Hao,2 Li-Hong Yao,2 Zhi-Ping Xie,2 HanChun Gao,2 Le-Yun Xie,3 Li-li Zhong,3 Bing Zhang,3 You-De Cao1 and Zhao-Jun Duan2 1 Department of Laboratory Medical, The First Affiliated Hospital of Hunan Normal University,Changsha, 2Department of Viral Diarrhoea, National Institute for Viral Disease Control and Prevention, CDC China, Beijing, and 3Department of Paediatrics, The First Affiliated Hospital of Hunan Normal University, Changsha, China
doi:10.1111/imm.12202 Received 25 August 2013; revised 30 September 2013; accepted 15 October 2013. Correspondence: Zhao-jun Duan, Department of Viral Diarrhoea, National Institute for Viral Disease Control and Prevention, CDC China, 100 Ying-Xin Sreet, Xuan-Wu District, Beijing 100052, China. Email: [email protected] and You-De Cao, Department of Laboratory Medical, The First Affiliated Hospital of Hunan Normal University, 61 Jiefang West Road, Changsha, Hunan Province, 410005 China. Email: [email protected] Senior author: Zhao-Jun Duan
Summary Human bocavirus (HBoV), a recently identified pathogen with a worldwide distribution is closely related to paediatric acute respiratory infection and gastroenteritis. The present study was performed to evaluate the immunogenicity of HBoV1 and HBoV2 virus-like particles (VLPs) as vaccine candidates in mice. Both HBoV1 and HBoV2 VLPs were expressed in the bacmid virus–SF9 cell system. Mice were inoculated three times at 3week intervals with HBoV VLPs at one dose intramuscular (i.m.) or intradermal (i.d.) with or without the addition of the alum adjuvant. ELISA was used to detected antibody, and ELISPOT was used to test cellular immune responses. HBoV-specific IgG antibodies were induced and alum adjuvant improved the antibody titres and avidity, while the inoculation pathway had no influence. T helper type 1/ type 2 immune responses were balanced induced by HBoV1 VLPs but not HBoV2 VLPs. Serum IgG antibody cross-reactivity rates of the two subtypes were similar, but crossreactions of HBoV1 immunization groups were higher. The single i.m. group had more interferon-c-secreting splenocytes. These data indicate that HBoV VP2 VLPs have good immunogenicity with induction of strong humoral and cellular immune responses, and they may be potential candidate vaccines for HBoV infection. Keywords: human bocavirus; immunogenicity; virus-like particle; VP2.
Introduction In 2005, Allander et al.1 reported the discovery of a new virus in paediatric respiratory nasopharyngeal aspirates, which was named human bocavirus (HBoV) and was shown to be a member of the parvovirus family. Three additional species, designated HBoV2–4, were identified subsequently.2 Previous studies have shown that HBoV1 is a causative agent of respiratory symptoms in children and adults;3–6 and HBoV2 and probably also HBoV3 were related to gastroenteritis but HBoV2 was predominant.2 Two recent studies indicate that HBoV is associated with interstitial pneumonia7 or even some lung and colorectal cancers and that it persists in solid tumours.8 To date, there have been no comparative studies of antiviral agents 58
and methods for prevention of HBoV infection.2 It is necessary to develop an appropriate vaccine. Virus-like particles (VLPs) have the external appearance of the authentic virion but no self-replication capability in cells, and can therefore elicit strong humoral immunity.9 Furthermore, VLPs can be processed and presented by MHC class I molecules to cytotoxic T lymphocytes, and elicit a cellular immune response.9,10 Many studies have confirmed that VLPs can be used as safe and effective vaccines.9,11 VLP vaccines for human papillomavirus12 and hepatitis B virus13 have been developed commercially, and confirmed to be highly effective against the corresponding diseases.14,15 Human bocavirus is a linear single-stranded DNA virus belonging to the family Parvoviridae, with a genome of ª 2013 John Wiley & Sons Ltd, Immunology, 142, 58–66
Immunogenicity of HBoV-1,2 VP2 VLPs in mice only approximately 5 kb in length.2 The virus possesses two main structural proteins, VP1 and VP2, which are both important components of the virus capsid.2 Bernstein et al.16 confirmed the immunogenicity of parvovirus B19 VP1 and VP2 VLPs. Their results indicated that VP1 plus VP2 VLPs induced antibodies that neutralized viral infectivity. However, Kantola et al.17 reported that HBoV VP2 was superior to the unique part of VP1 with respect to immunoreactivity. Furthermore, most anti-HBoV antibodies in human serum seem to anchor the viral capsid protein VP2.18 These results suggest that VP2 is a more appropriate HBoV vaccine candidate than VP1. Moreover, HBoV VP2 has been successfully expressed as VLPs, which induced specific antibody in rabbits.19–21 However, there have been no studies regarding the immunogenicity of VP2. In the present study, we expressed HBoV1 and HBoV2 VP2 VLPs and evaluated their immunogenicity according to different routes of immunization [intramuscular (i.m.) and intradermal (i.d.)] with or without alum adjuvant in mice. The specific IgG titres, antibody avidity, IgG1/IgG2a cross-reactivity and specific cellular immune responses were investigated.
Materials and methods HBoV virus-like particle preparation The VP2 gene sequences of HBoV1 and HBoV2 were obtained from GenBank (2010, GenBank ID: NC_007455;2009, GenBank ID: NC_012042). The codons were optimized based on the codon preference of SF9 insect cells using the GENSCRIPT OPTIMUMGENETM software (GenScript, NJ). The genes of the two VP2 proteins were synthesized by Invitrogen (Carlsbad, CA), expressed in the bacmid virus–SF9 cell system (Invitrogen), and purified with caesium chloride (Alfa Aesar, Tianjin, China) discontinuous density gradient centrifugation as described previously.22 The VLPs were identified by SDS–PAGE and Western blot. The structures of purified VLPs were visualized by transmission electron microscopy (Tecnai 12; FEI, Blackwood, NJ).Concentrations of the purified VLPs were determined using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL).
Animal studies BALB/c mice (male, 6–8 weeks old) were purchased from Vital River Laboratories (Beijing, China) and housed in the Animal Laboratory of China CDC. Animal experiments were approved by the Beijing Association for Laboratory Animal Care. Immunization of mice. Experimental animals (10 mice per group) were immunized by i.m. (hind thigh) or i.d. (skin of abdomen) injection with purified VLPs or ª 2013 John Wiley & Sons Ltd, Immunology, 142, 58–66
VLPs + Imject Alum Adjuvant (Thermo Scientific, Waltham, MA). Each mouse was immunized with 15 lg of VLPs diluted in 100 ll PBS on study weeks 0, 3 and 6. Animals in the control group (five mice per group) were administered PBS without VLPs by the same route and according to the same schedule. Blood sample collection. Blood samples were obtained from the tail vein on study weeks 0 (before primary immunization), 2, 4, 6 and 8. All mice were killed on study week 8, and whole blood and spleen tissue were collected.
Serum and splenic lymphocyte preparation Blood samples were centrifuged (Eppendorf, Hamburg, Germany) at 2600 g for 10 min after cooling by storage at 4° for 4–6 hr. The serum was then removed and stored at 20°. Mouse spleen tissues were separated in aseptic conditions, and collected in mouse splenocyte separation medium (Dakewei biotech, Beijng, Biotech, Beijing, China), the structure of the spleen was disrupted using a disposable sterile syringe, and the destroyed issue was filtered using 70-ll cell strainers (BD Falcon, Franklin Lake, NJ). Cell suspensions were centrifuged at 800 g for 30 min (5804R; Eppendorf), to obtain single lymphocytes. After washing once with RPMI-1640 medium (Gibco, Grand Island, NY), The single lymphocytes were resuspended in complete medium consisting of RPMI1640 medium supplemented with 10% fetal calf serum, 1% penicillin and streptomycin, and 1% L-glutamine (Gibco).
Peptide design, synthesis and verification Mouse T-cell epitopes of HBoV VP2 have not yet been reported. So synthetic peptides corresponding to the mouse T-cell epitopes of HBoV VP2 used in ELISPOT assays as specific stimuli were predicted and verified as described previously.23,24 According to the amino acid sequence of the targeted protein, these potential T-cell epitopes were predicted by using computer simulation of the possible spatial structure of polypeptide. Briefly, the whole amino acid sequences of HBoV1 and HBoV2 VP2 were submitted to SYFPEITHI (http://www.syfpeithi.de) and NetMHC 3.2 server (http://www.cbs.dtu.dk/services/ NetMHC). In each genotype, five peptides (two for 15-mers and three for 9-mers) were selected by their scores from high to low in the prediction software and then synthesized by SciLight Peptide (Beijing, China). Peptides were dissolved in RPMI-1640 medium and diluted to the working concentration of 20 lg/ml in complete medium and stored at 20° until further use. ELISPOT assay was performed to identify effective specific peptides. 59
Z.-H. Deng et al. IgG and IgG subtype ELISA The ELISA operation steps were described previously.21 The end-point titres are reported as the highest dilution at which the optical density at 450 nm (OD450) was 21-fold higher than that of the negative control serum.
Specific IgG avidity assay The antibody avidity assay was performed as described previously.25,26 The steps involved were the same as for the IgG and IgG subtype ELISAs, except that after discarding the serum (1 : 200 dilution), 8 M urea (Promega, Madison, WI) was added to wells (200 ll/well) followed by incubation for 5 min at room temperature; this procedure was repeated once to separate the low-activity antibody from the antigen–antibody complex. The avidity index (AI) was calculated as follows: AI ¼ ðOD450 with ureaÞ=ðOD450 without ureaÞ 100% The cut-off for judging the avidity was 50%.
labelled anti-mouse IFN-c antibody (BD Biosciences) was added and incubated for 2 hr at room temperature. After washing three times with PBS-T, horseradish peroxidase-labelled streptavidin was added at a dilution of 1 : 100, and incubated for 1 hr at room temperature. After washing, the spots were developed with a 3-amino9-ethylcarbazole substrate set (BD Biosciences). Spots were counted with a Bioreader (Biosys, Heidelberg, Germany).
Statistical analysis The Mann–Whitney U-test was used to compare the serum IgG titres and analysis of variance (ANOVA) was used to compare the IgG avidities, IgG1/IgG2a titres, and cellular immune responses determined by IFN-c ELISPOT assay in both HBoV1 and HBoV2 immunization groups. Student’s t-test was used to evaluate differences in serum IgG titres, IgG avidities and cellular immune responses determined by IFN-c ELISPOT assay. Statistical analyses were performed using SPSS ver. 17.0 (SPSS, Chicago, IL). In all analyses, P < 005 was taken to indicate statistical significance.
Cross-reaction and cross-reaction avidity assay The assay was based on the ELISAs described above. Sera were collected from mice immunized with HBoV1 or HBoV2 VLPs on study week 8, divided into three equal portions, and diluted 1 : 200 with PBS-T. Two portions were added to 96-well microplates coated with HBoV1 or HBoV2 VLPs, and the third was used for avidity assay and added to microplates coated with HBoV1 or HBoV2 VLPs. The following steps were identical to those of the IgG and IgG subtype ELISAs or specificity IgG avidity assay described above. The cross-reaction rate (CRR) was calculated as follows: CRR ¼ ðOD450 other-VLPs/OD450 self-VLPsÞ 100% The cross-reaction avidity index was calculated as described above.
ELISPOT interferon-c assay Ninety-six-well ELISPOT plates (BD Biosciences, San Diego, CA) were coated at 4° overnight with 05 lg unlabelled mouse interferon-c (IFN-c) antibody (BD Biosciences) and blocked by incubation for 2 hr at room temperature with blocking solution. Splenocytes (5 9 105) were added to each well, and cultivated with HBoV1-specific or HBoV2-specific peptide for 24 hr at 37° in a 5% CO2 atmosphere. Culture medium containing 10 lg/ml concanavalin A (Con A) (Invitrogen) without peptide was used as the control. After aspiration of the cell suspension, wells were washed first with deionized water and then with PBS-T. Then, 02 µg biotin-
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Results Characteristics of HBoV1 and HBoV2 VLPs The molecular weights of high-purity HBoV1 and HBoV2 VLPs were approximately 60 000 (Fig. 1a,b), consistent with previous reports.20,21 On transmission electron microscopy, the VLPs were shown to be approximately 21–24 nm in diameter (Fig. 1c), which is similar to human bocavirus and other parvoviruses reported previously.27,28 The HBoV1 and HBoV2 VLP concentrations determined by BCA protein assay were 1816 and 2964 lg/ml, respectively (data not shown).
A high level of HBoV-specific IgG was induced by HBoV VLPs Administration (i.m. and i.d.) of both HBoV1 (Fig. 2a) and HBoV2 (Fig. 2b) VLPs with alum induced strong HBoV-specific IgG responses in all of the experimental animals. The serum IgG titres of mice at week 8 were subjected to logarithmic transformation and compared (Fig. 2c). There were no differences between the two immunization routes for both HBoV1 and HBoV2 (P > 005), but VLP immunization with alum induced significantly higher IgG titres than VLP immunization alone with both immunogens (P < 001). The IgG titre induced by immunization with HBoV1 VLPs was higher than that induced by immunization with HBoV2 (P < 001).
ª 2013 John Wiley & Sons Ltd, Immunology, 142, 58–66
Immunogenicity of HBoV-1,2 VP2 VLPs in mice (a)
M
HBoV1
HBoV2
(b)
HBoV1
HBoV2 Marker ~175 000 ~130 000 ~95 000 ~70 000
90 000
~62 000
66 000
~51 000 ~42 000
45 000 ~29 000 ~22 000
35 000
~14 000 29 000
~10 500
SDS–PAGE
Figure 1. The purity and molecular weight of human bocavirus type 1 (HBoV1) and HBoV2 VP2 virus-like particles (VLPs) with molecular weight approximately 60 000 used to immunize mice and as antigen in ELISA are shown (15% SDS–PAGE) (a). The specificities of HBoV1 and HBoV2 VP2 VLPs were determined by Western blotting (b). Structures of VLPs were observed by transmission electron microscopy. The diameter was approximately 21–24 nm (c).
Western blot
(c)
100 nm
X97000 HBoV1 VLPs
100 nm
X97000 HBoV2 VLPs
Serum HBoV-specific IgG activity induced by VLP immunization was higher with than without alum adjuvant
ity index than HBoV2-immunized serum (P < 001) (Fig. 4).
The mean avidity indexes of each group are shown in Fig. 3. The IgG avidities induced by immunization with HBoV1 and HBoV2 VLPs through both i.m. and i.d. routes were higher with alum adjuvant than without alum adjuvant. The ANOVA indicated that VLPs injected in conjunction with alum induced higher IgG avidity than VLPs administered alone for both HBoV1 (P < 001) and HBoV2 (P < 001), but there were no differences related to immunization route (i.m. versus i.d.) (P > 005) for both HBoV1 and HBoV2. A Student’s t-test indicated no differences in the IgG avidities induced by HBoV1 and HBoV2 VLPs (P > 005).
Increased cellular immunity was induced by single immunization with VLPs via i.m. injection
Increased cross-reaction avidity was induced by HBoV1 VLPs The cross-reaction rates in HBoV1-immunized serum reacting with HBoV2 VLPs and HBoV2-immunized serum showed no difference between the two subtypes (P > 005). However, the cross-reaction avidities induced by HBoV1 VLPs and HBoV2 VLPs differed; HBoV1 VLP-immunized serum had a higher cross-reaction avidª 2013 John Wiley & Sons Ltd, Immunology, 142, 58–66
The T-cell epitopes of HBoV were predicted (Table 1) and verified by ELISPOT assay Two nine-amino-acid HBoV1-specific epitopes (P3:GYIPIENEL; P5:LYQMPFFLL) and one nine-amino-acid HBoV2-specific epitope (P8:GYIVIHEL) showed high activity and specificity but little cross-reactivity. P3 and P8 were selected as the HBoV1- and HBoV2-specific epitopes, respectively, for the ELISPOT assay. The cellular immune response was then evaluated by ELISPOT assay (Fig. 5). In contrast to the serum IgG antibody response, i.m. and i.d. immunization with VLPs alone induced higher levels of cellular immunity for both HBoV1 and HBoV2 compared with immunization in conjunction with alum adjuvant (i.m., P < 0001; i.d., P < 001). The immunization route, whether i.m. or i.d., did not affect the response induced by HBoV1 VLPs with or without alum (P > 005), or that induced by HBoV2 VLPs with alum adjuvant (P > 005). However, the route of immunization with HBoV2 VLPs without
61
Z.-H. Deng et al. HBoV1 VLPs Immunization
(a) Antibody titre (log2)
22 20
VLPs i.m. VLPs+Alum i.m. VLPs i.d. VLPs+Alum i.d.
18 16 14
2
4
6
8
Time (weeks) HBoV2 VLPs Immunization
(b) Antibody titre (log2)
22 20
VLPs i.m. VLPs+Alum i.m. VLPs i.d. VLPs+Alum i.d.
18 16 14
2
4
6
8
Time (weeks) HBoV VLP– induced IgG response
(c)
** 22
**
**
***
IgG titre (log2)
** 20
VLPs i.m. VLPs+Alum i.m. VLPs i.d. VLPs+Alum i.d.
18 16 14
HBoV1 VLPs
HBoV2 VLPs Immunogens
Figure 2. Antibody titres are shown as geometric means. Serum-specific IgG titres increased over time (a, b). Serum IgG titres collected on study week 8 were logarithmically transformed, and the Mann–Whitney U-test was used for comparison between immunization via intradermal (i.d.) and intramuscular (i.m.) routes with or without alum adjuvant. The Student’s t-test was used to compare the IgG titres induced between human bocavirus type 1 (HBoV1) and HBoV2 virus-like particles (VLPs) (c). *P < 005, ** P < 001,*** P < 0001.
HBoV VLP–induced IgG Avidity
Avidity index (%)
100
*
*
*
*
80
VLP i.m. VLP i.d. VLPs+Alum i.m. VLP+Alum i.d.
60 40 20 0
HBoV1
HBoV2 Immunogen
Figure 3. Human bocavirus (HBoV) -specific IgG avidities are shown as the means and standard deviation of avidity index (AI). Sera were collected from mice on study week 8 and diluted 1 : 200 in PBS-T. The AI was calculated as follows[AI = (OD450 with urea)/(OD450 without urea) 9 100%],analysis of variance was used to compare the IgG avidity indexes between immunization via intradermal (i.d.) and intramuscular (i.m.) routes with and without alum adjuvant The Student’s t-test was used to compare the IgG avidity index between HBoV1 and HBoV2 viruslike particles (VLPs). *P < 005.
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ª 2013 John Wiley & Sons Ltd, Immunology, 142, 58–66
Immunogenicity of HBoV-1,2 VP2 VLPs in mice Cross-reaction rate
Cross-reaction avidity index
100
(1 : 290 000) (P < 005), and was also unaffected by the presence or absence of alum adjuvant (data not shown).
**
Rate (%)
80
Discussion
60
40
20
0
HBoV1
HBoV2 Immunogen
Figure 4. Cross-reaction rate was used to describe the proportions of human bocavirus type 2 (HBoV2) and HBoV1 virus-like particle (VLP) combined IgG antibodies in the sera of HBoV1 and HBoV2 VLP-immunized mice, respectively. A cross-reaction index was used to describe the activities of these cross-reactive IgG antibodies. *P < 005, **P < 001 (Student’s t-test). Table 1. List of synthesized peptides Be form
Start position
Amino acid sequence
Number
HBoV1 VP2
260 46 191 130 217 258 120 193 49 187
GLMFNPKVPTRRVQY GSHFSDKYVVTKNTR GYIPIENEL IYNLQIKQI LYQMPFFLL GLMFNPKVPTRRAQY YKRFKPRKMHVKIYN GYIPVIHEL SYFTDSYVI WYLFQYGYI
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10
HBoV2 VP2
The predicted human bocavirus (HBoV) -specific peptides are shown. Five peptides were selected based on higher scores and water solubility for each subtype of HBoV. The start positions of each peptide in the viral protein (VP2) amino acid sequence are shown together with peptide number. Be form means that peptides from which genotype of HBoV.
adjuvant affected (P < 001).
the
IFN-c
response
significantly
The T helper type 1/type 2 immune response induced by VLPs with or without adjuvant The T helper type 1 (Th1)/Th2 type immune responses induced by HBoV VLPs were determined (Fig. 6). The IgG2a and IgG1 antibody titres were logarithmically transformed and compared. HBoV1 VLPs induced a balanced IgG1 and IgG2a response (P > 005), which was not affected by the presence or absence of alum adjuvant. However, HBoV2 VLPs induced a twofold higher IgG1 antibody level (1 : 540 000) compared with that of IgG2a ª 2013 John Wiley & Sons Ltd, Immunology, 142, 58–66
In the present study, HBoV VLPs induced high levels of serum IgG, as reported previously for other VLPs.29 This is because VLPs can efficiently combine with the B-cell receptor, which consists of immunoglobulin molecules, and the interaction between B cells and VLPs induces high levels of neutralizing antibodies.9 Furthermore, the HBoV VLPs induced high titres of IgG antibodies in mice without any adjuvant, which is in accordance with some previous studies.30,31 However, higher IgG titres were induced by immunization with HBoV VLPs with alum adjuvant. Alum may attract antigen-presenting cells to the injection site to recognize and engulf the antigens.32,33 In addition, alum adjuvant was reported to absorb antigens and form an antigen library to continuously stimulate immunocytes.34,35 Therefore, vaccines administered with alum adjuvant can induce higher levels of humoral immunity. These results suggest that HBoV VP2 VLPs could be administered in conjunction with alum adjuvant to improve immunogenicity. High levels of IgG do not necessarily correspond to a high rate of clearance of exogenous virus. However, antibody avidity plays an important role, as high-avidity antibodies bind strongly to exogenous virus. In the present study, i.m. and i.d. immunization with HBoV1 and HBoV2 VLPs with or without alum adjuvant resulted in high IgG avidity. Furthermore, there were no differences in antibody avidity between the two HBoV genotypes, as determined by ANOVA. However, alum adjuvant markedly increased serum IgG avidity, whereas immunization route had no such influence. These results suggest that serum IgG induced by immunization with HBoV VLPs and alum adjuvant more effectively clear exogenous virus and protect against infection, so alum should be used in HBoV VLP vaccination. A previous study indicated serum cross-reactivity between HBoV1 and HBoV2–4,20 which may be a result of the high degree of VP2 gene homology among these viruses. In the present study, the rates of cross-reaction of HBoV1-immunized serum with HBoV2 VLPs and HBoV2-immunized serum with HBoV1 VLPs were 743 96% and 698 152%, respectively. These differences in cross-reactivity were not significant (P > 005). However, the serum avidity cross-reaction rate differed between sera from mice that received immunization with HBoV1 and HBoV2 VLPs. The serum IgG induced by HBoV1 VLPs showed high avidity with HBoV2 VLPs, with an avidity index of 564 120%, which was significantly higher than that of HBoV2immunized serum (326 92%) (P < 001). Cross-neutralizing antibodies can mediate cross-protection.36 These 63
Z.-H. Deng et al. (a)
HBoV1 VLP-induced cellular immune response 250
***
Spots/106 cells
200
***
150 100 50 0 VLPs IM VLPs+Alum VLPs ID VLPs+Alum PBS IM PBS+Alum PBS ID PBS+Alum i.m. i.d. i.m. i.d. Experimental groups
(b)
** 250
HBoV2 VLP-induced cellular immune response
***
Spots/106 cells
200 150 ** 100 50 0 VLPs IM VLPs+Alum VLPs ID VLPs+Alum PBS IM PBS+Alum PBS ID PBS+Alum ID IM IM ID Experimental groups
results suggest that HBoV1 VLPs would provide better protection than HBoV2 VLPs when administered at appropriate doses. However, we did not address crossreactivity between HBoV1–2 VLPs and HBoV3 and HBoV4. Characterization of the cellular immune response is an indispensable part of the evaluation of a candidate vaccine. Measurement of the IFN-c level is often used to evaluate the cellular immune response. In the present study, IFN-c production was induced in the splenocytes of mice immunized with HBoV1 and HBoV2 VLPs. These data confirm that both HBoV1 and HBoV2 VLPs can activate T cells and stimulate cellular immunity. Statistical analysis indicated that alum adjuvant had the opposite effect on the cellular immune response. This is contrary to what it does in the humoral immune response. That is, the cellular immune response, as monitored by IFN-c ELISPOT assay, was induced more strongly by immunization with VLPs alone compared with by immunization with VLPs plus alum adjuvant. It is known that aluminium adjuvants can advantageously regulate lymphocyte differentiation into helper T lymphocytes and enhance the humoral immune response, but weaken cytotoxic T lymphocyte differentiation and prolif64
Figure 5. Production of interferon-c (IFN-c) as a cellular immunity-associated cytokine was determined by ELISPOT assay. The effective human bocavirus (HBoV) -specific peptides were selected from Table 1, HBoV1-specific P3 (GYIPIENEL) and HBoV1-specific P8 (GYIPVIHEL) were used as specificity stimuli. The ELISPOT results were converted into spots/106 cells. The results for each group are shown as the means and standard deviation (a, b). Analysis of variance was used to compare the spots between immunization via intradermal (i.d.) and intramuscular (i.m.) routes with or without alum adjuvant. *P < 005, **P < 001, ***P < 0001.
eration.37 This seemed to be because the antigen library established by injection with alum adjuvant prolongs the stimulation, but also reduces the intensity of stimulus at the same time, and the presence of alum did not improve the cellular immune response.32,38 The cellular immune responses of the two genotypes were not compared because of the different specific stimulus peptides. However, there have been no previous studies of the T-cell epitopes of HBoV in mice. The sequences of stimulus peptides used in the present study were predicted using a bioinformatics method described previously.23,24 Our results confirmed that HBoV VLPs induced a specific cellular immune response, but do not reflect the entire cellular immune response in vivo, and further studies that compare all possible T-cell epitopes are required. Detection of IgG1 and IgG2a is usually performed to determine whether vaccine-elicited immunity tends toward the Th1 or Th2 type. In mice, the Th1-type immune response produces IgG2a subtype antibodies, whereas the Th2-type immune response produces IgG1 subtype antibodies. However, the immune response in humans is the reverse; i.e. IgG1 reflects a Th1-type immune response, while IgG2a reflects a Th2-type immune response.29,39,40 Some previous reports indicate ª 2013 John Wiley & Sons Ltd, Immunology, 142, 58–66
Immunogenicity of HBoV-1,2 VP2 VLPs in mice (a)
HBoV1 VLP immunization 3 VLP-induced IgG1 VLP-induced IgG2a
2·5
VLP+Alum-induced IgG1 VLP+Alum-induced IgG2a
OD 450
2
PBS control group mice IgG1 PBS control group mice IgG2a
1·5
PBS+Alum control group mice IgG1 PBS+Alum control group mice IgG2a
1 0·5
·8 25 ·6 51 ·2 10 2· 4 20 4· 8 40 9· 6 81 9· 2 16 38 · 32 4 76 ·8
4
12
2
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6
3·
8
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4
0·
2 0·
0·
1 0·
0 Serum dilution (×1000)
HBoV2 VLP immunization
(b) 3
VLP-induced IgG1 VLP-induced IgG2a VLP+Alum-induced IgG1 VLP+Alum-induced IgG2a PBS control group mice IgG1 PBS control group mice IgG2a PBS+Alum control group mice IgG1 PBS+Alum control group mice IgG2a
2·5
OD 450
2 1·5 1 0·5
that VLPs usually induce a balanced Th1/Th2 immune response;29 this was indeed found in the present study. The end-point titres of IgG1/IgG2a in serum induced by HBoV VLP immunization were determined by ELISA. The results indicated that HBoV1 VLPs induced a balance of Th1/Th2 immune response, reflected by high but not significantly different serum IgG2a/IgG1 antibody titres. However, the immune response induced by HBoV2 VLPs was different from that induced by HBoV1 VLPs. That is, the serum IgG1 titre induced by HBoV2 VLPs was higher than the serum IgG2a antibody titre. This suggests that HBoV2 VLPs induce a stronger IgG1-subtype immune response than IgG2a-subtype immune response, which is indicative of a stronger Th2-type than Th1-type immune response. In conclusion, our data show that HBoV VP2 VLPs have good immunogenicity and induce strong humoral and cellular immune responses in mice. Therefore, HBoV VP2 VLPs represent good candidate proteins for HBoV vaccine. It should be noted that we did not determine the protective efficacy of this VLP and did not perform a neutralization test without live virus and HBoV-sensitive animals or cells, which are essential in a neutralization test. Recently, successful production of the HBoV1 virons in vitro through transfection of a plasmid-like HBoV into human embryonic kidney 293 cells has been reported and ª 2013 John Wiley & Sons Ltd, Immunology, 142, 58–66
·4 76 ·8 32
2
38
16
6
81
9·
8
9· 40
4
4·
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·2
·6
2· 10
51
25
·8 12
4 6·
2 3·
6 1·
8 0·
4 0·
2 0·
1
0
0·
Figure 6. Sera were collected from mice on study week 8. The results of IgG1 and IgG2a subtype antibodies are shown as means of OD450 from ELISA. Serum dilutions were doubled starting from 1 : 100.
Serum dilution (×1000)
these virions are able to infect human airway epithelial cells,41 which will promote HBoV vaccine development.
Acknowledgements We are grateful to Baoying Huang and Jingdong Song for technical assistance during the study. This work was partly supported by a research conditions innovation project grant (2012TT2004) from the Science and Technology Agency of Hunan Province, China.
Disclosures None of the authors has a conflict of interest.
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23 Rammensee H, Bachmann J, Emmerich NP, Bachor OA, Stevanovic S. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 1999; 50:213–9. 24 Lundegaard C, Lamberth K, Harndahl M, Buus S, Lund O, Nielsen M. NetMHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8–11. Nucleic Acids Res 2008; 36(Web Server issue):W509–12. 25 Wilson KM, Di Camillo C, Doughty L, Dax EM. Humoral immune response to pri-
Virol 2013; 58:325–7. 8 Schildgen V, Malecki M, Tillmann RL, Brockmann M, Schildgen O. The human bocavirus is associated with some lung and colorectal cancers and persists in solid tumors. PLoS One 2013; 8:e68020. 9 Chackerian B. Virus-like particles: flexible platforms for vaccine development. Expert Rev Vaccines 2007; 6:381–90.
mary rubella virus infection. Clin Vaccine Immunol 2006; 13:380–6. 26 Nedeljkovic J, Jovanovic T, Oker-Blom C. Maturation of IgG avidity to individual rubella virus structural proteins. J Clin Virol 2001; 22:47–54. 27 Gurda BL, Parent KN, Bladek H et al. Human bocavirus capsid structure: insights into the structural repertoire of the parvoviridae. J Virol 2010; 84:5880–9. 28 Brieu N, Gay B, Segondy M, Foulongne V. Electron microscopy observation of human
10 Ludwig C, Wagner R. Virus-like particles – universal molecular toolboxes. Curr Opin Biotechnol 2007; 18:537–45. 11 Roldao A, Mellado MC, Castilho LR, Carrondo MJ, Alves PM. Virus-like particles in vaccine development. Expert Rev Vaccines 2010; 9:1149–76. 12 Paavonen J, Jenkins D, Bosch FX et al. Efficacy of a prophylactic adjuvanted bivalent L1 virus-like-particle vaccine against infection with human papillomavirus types 16 and 18 in young women: an interim analysis of a phase III double-blind, randomised con-
bocavirus (HBoV) in nasopharyngeal samples from HBoV-infected children. J Clin Microbiol 2007; 45:3419–20. 29 Tamminen K, Huhti L, Koho T, Lappalainen S, Hytonen VP, Vesikari T, Blazevic V. A comparison of immunogenicity of norovirus GII-4 virus-like particles and P-particles. Immunology 2011; 135:89–99. 30 Buonaguro L, Tornesello ML, Buonaguro FM. Virus-like particles as particulate vaccines. Curr HIV Res 2010; 8:299–309.
trolled trial. Lancet 2007; 369:2161–70. 13 Kee GS, Pujar NS, Titchener-Hooker NJ. Study of detergent-mediated liberation of hepatitis B virus-like particles from S. cerevisiae homogenate: identifying a framework for the design of future-generation lipoprotein vaccine processes. Biotechnol Prog 2008; 24:623–31. 14 Inoue M. Protection against uterine cervical cancer by HPV vaccines. Uirusu 2008;
31 Zhang LF, Zhou J, Chen S et al. HPV6b virus like particles are potent immunogens without adjuvant in man. Vaccine 2000; 18:1051–8. 32 McKee AS, Munks MW, MacLeod MK, Fleenor CJ, Van Rooijen N, Kappler JW, Marrack P. Alum induces innate immune responses through macrophage and mast cell sensors, but these sensors are not required for alum to act as an adjuvant for specific immunity. J Immunol 2009; 183:4403–14.
58:155–63. 15 Stanley M, Gissmann L, Nardelli-Haefliger D. Immunobiology of human papillomavirus infection and vaccination – implications for second generation vaccines. Vaccine 2008; 26(Suppl. 10):K62–7. 16 Bernstein DI, El Sahly HM, Keitel WA et al. Safety and immunogenicity of a candidate parvovirus B19 vaccine. Vaccine 2011; 29:7357–63.
33 Calabro S, Tortoli M, Baudner BC et al. Vaccine adjuvants alum and MF59 induce rapid recruitment of neutrophils and monocytes that participate in antigen transport to draining lymph nodes. Vaccine 2011; 29:1812–23. 34 Lambrecht BN, Kool M, Willart MA, Hammad H. Mechanism of action of clinically approved adjuvants. Curr Opin Immunol 2009; 21:23–9. 35 Crisci E, Fraile L, Moreno N et al. Chimeric calicivirus-like particles elicit specific
17 Kantola K, Hedman L, Allander T, Jartti T, Lehtinen P, Ruuskanen O, Hedman K, Soderlund-Venermo M. Serodiagnosis of human bocavirus infection. Clin Infect Dis 2008; 46:540–6. 18 Lindner J, Karalar L, Zehentmeier S et al. Humoral immune response against human bocavirus VP2 virus-like particles. Viral Immunol 2008; 21:443–9. 19 Kahn JS, Kesebir D, Cotmore SF, D’Abramo A Jr, Cosby C, Weibel C, Tattersall P. Seroepidemiology of human bocavirus defined using recombinant virus-like particles. J
immune responses in pigs. Vaccine 2012; 30:2427–39. 36 Kemp TJ, Hildesheim A, Safaeian M et al. HPV16/18 L1 VLP vaccine induces crossneutralizing antibodies that may mediate cross-protection. Vaccine 2011; 29:2011–4. 37 Fiejka M, Aleksandrowicz J. Aluminum as an adjuvant in vaccines and post-vaccine reactions. Rocz Panstw Zakl Hig 1993; 44:73–80. 38 Kool M, Fierens K, Lambrecht BN. Alum adjuvant: some of the tricks of the oldest adjuvant. J Med Microbiol 2012; 61(Pt 7):927–34.
Infect Dis 2008; 198:41–50. 20 Kantola K, Hedman L, Arthur J et al. Seroepidemiology of human bocaviruses 1–4. J Infect Dis 2011; 204:1403–12. 21 Lin F, Guan W, Cheng F, Yang N, Pintel D, Qiu J. ELISAs using human bocavirus VP2 virus-like particles for detection of antibodies against HBoV. J Virol Methods 2008; 149:110–7.
39 Ormstad H, Groeng EC, Duffort O, Lovik M. The effect of endotoxin on the production of IgE, IgG1 and IgG2a antibodies against the cat allergen Fel d 1 in mice. Toxicology 2003; 188:309–18. 40 Hollo K, Glant TT, Garzo M, Finnegan A, Mikecz K, Buzas E. Complex pattern of Th1 and Th2 activation with a preferential increase of autoreactive Th1 cells in BALB/c mice with proteoglycan (aggrecan)-induced arthritis. Clin Exp Immunol 2000; 120:167–73.
22 Zhao LQ, Qian Y, Ding YX, Zhu RN, Deng J, Wang F, Sun Y, Li Y. The expression of the capsid protein VP2 from human bocavirus identified in Beijing and the formation of virus-like particles (VLPs) in insect cells. Bing Du Xue Bao 2009; 25:333–8.
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ª 2013 John Wiley & Sons Ltd, Immunology, 142, 58–66
ORIGINAL ARTICLE
Immunogenicity of recombinant human bocavirus-1,2 VP2 gene virus-like particles in mice Zhong-Hua Deng,1,2 Ye-Xia Hao,2 Li-Hong Yao,2 Zhi-Ping Xie,2 HanChun Gao,2 Le-Yun Xie,3 Li-li Zhong,3 Bing Zhang,3 You-De Cao1 and Zhao-Jun Duan2 1 Department of Laboratory Medical, The First Affiliated Hospital of Hunan Normal University,Changsha, 2Department of Viral Diarrhoea, National Institute for Viral Disease Control and Prevention, CDC China, Beijing, and 3Department of Paediatrics, The First Affiliated Hospital of Hunan Normal University, Changsha, China
doi:10.1111/imm.12202 Received 25 August 2013; revised 30 September 2013; accepted 15 October 2013. Correspondence: Zhao-jun Duan, Department of Viral Diarrhoea, National Institute for Viral Disease Control and Prevention, CDC China, 100 Ying-Xin Sreet, Xuan-Wu District, Beijing 100052, China. Email: [email protected] and You-De Cao, Department of Laboratory Medical, The First Affiliated Hospital of Hunan Normal University, 61 Jiefang West Road, Changsha, Hunan Province, 410005 China. Email: [email protected] Senior author: Zhao-Jun Duan
Summary Human bocavirus (HBoV), a recently identified pathogen with a worldwide distribution is closely related to paediatric acute respiratory infection and gastroenteritis. The present study was performed to evaluate the immunogenicity of HBoV1 and HBoV2 virus-like particles (VLPs) as vaccine candidates in mice. Both HBoV1 and HBoV2 VLPs were expressed in the bacmid virus–SF9 cell system. Mice were inoculated three times at 3week intervals with HBoV VLPs at one dose intramuscular (i.m.) or intradermal (i.d.) with or without the addition of the alum adjuvant. ELISA was used to detected antibody, and ELISPOT was used to test cellular immune responses. HBoV-specific IgG antibodies were induced and alum adjuvant improved the antibody titres and avidity, while the inoculation pathway had no influence. T helper type 1/ type 2 immune responses were balanced induced by HBoV1 VLPs but not HBoV2 VLPs. Serum IgG antibody cross-reactivity rates of the two subtypes were similar, but crossreactions of HBoV1 immunization groups were higher. The single i.m. group had more interferon-c-secreting splenocytes. These data indicate that HBoV VP2 VLPs have good immunogenicity with induction of strong humoral and cellular immune responses, and they may be potential candidate vaccines for HBoV infection. Keywords: human bocavirus; immunogenicity; virus-like particle; VP2.
Introduction In 2005, Allander et al.1 reported the discovery of a new virus in paediatric respiratory nasopharyngeal aspirates, which was named human bocavirus (HBoV) and was shown to be a member of the parvovirus family. Three additional species, designated HBoV2–4, were identified subsequently.2 Previous studies have shown that HBoV1 is a causative agent of respiratory symptoms in children and adults;3–6 and HBoV2 and probably also HBoV3 were related to gastroenteritis but HBoV2 was predominant.2 Two recent studies indicate that HBoV is associated with interstitial pneumonia7 or even some lung and colorectal cancers and that it persists in solid tumours.8 To date, there have been no comparative studies of antiviral agents 58
and methods for prevention of HBoV infection.2 It is necessary to develop an appropriate vaccine. Virus-like particles (VLPs) have the external appearance of the authentic virion but no self-replication capability in cells, and can therefore elicit strong humoral immunity.9 Furthermore, VLPs can be processed and presented by MHC class I molecules to cytotoxic T lymphocytes, and elicit a cellular immune response.9,10 Many studies have confirmed that VLPs can be used as safe and effective vaccines.9,11 VLP vaccines for human papillomavirus12 and hepatitis B virus13 have been developed commercially, and confirmed to be highly effective against the corresponding diseases.14,15 Human bocavirus is a linear single-stranded DNA virus belonging to the family Parvoviridae, with a genome of ª 2013 John Wiley & Sons Ltd, Immunology, 142, 58–66
Immunogenicity of HBoV-1,2 VP2 VLPs in mice only approximately 5 kb in length.2 The virus possesses two main structural proteins, VP1 and VP2, which are both important components of the virus capsid.2 Bernstein et al.16 confirmed the immunogenicity of parvovirus B19 VP1 and VP2 VLPs. Their results indicated that VP1 plus VP2 VLPs induced antibodies that neutralized viral infectivity. However, Kantola et al.17 reported that HBoV VP2 was superior to the unique part of VP1 with respect to immunoreactivity. Furthermore, most anti-HBoV antibodies in human serum seem to anchor the viral capsid protein VP2.18 These results suggest that VP2 is a more appropriate HBoV vaccine candidate than VP1. Moreover, HBoV VP2 has been successfully expressed as VLPs, which induced specific antibody in rabbits.19–21 However, there have been no studies regarding the immunogenicity of VP2. In the present study, we expressed HBoV1 and HBoV2 VP2 VLPs and evaluated their immunogenicity according to different routes of immunization [intramuscular (i.m.) and intradermal (i.d.)] with or without alum adjuvant in mice. The specific IgG titres, antibody avidity, IgG1/IgG2a cross-reactivity and specific cellular immune responses were investigated.
Materials and methods HBoV virus-like particle preparation The VP2 gene sequences of HBoV1 and HBoV2 were obtained from GenBank (2010, GenBank ID: NC_007455;2009, GenBank ID: NC_012042). The codons were optimized based on the codon preference of SF9 insect cells using the GENSCRIPT OPTIMUMGENETM software (GenScript, NJ). The genes of the two VP2 proteins were synthesized by Invitrogen (Carlsbad, CA), expressed in the bacmid virus–SF9 cell system (Invitrogen), and purified with caesium chloride (Alfa Aesar, Tianjin, China) discontinuous density gradient centrifugation as described previously.22 The VLPs were identified by SDS–PAGE and Western blot. The structures of purified VLPs were visualized by transmission electron microscopy (Tecnai 12; FEI, Blackwood, NJ).Concentrations of the purified VLPs were determined using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL).
Animal studies BALB/c mice (male, 6–8 weeks old) were purchased from Vital River Laboratories (Beijing, China) and housed in the Animal Laboratory of China CDC. Animal experiments were approved by the Beijing Association for Laboratory Animal Care. Immunization of mice. Experimental animals (10 mice per group) were immunized by i.m. (hind thigh) or i.d. (skin of abdomen) injection with purified VLPs or ª 2013 John Wiley & Sons Ltd, Immunology, 142, 58–66
VLPs + Imject Alum Adjuvant (Thermo Scientific, Waltham, MA). Each mouse was immunized with 15 lg of VLPs diluted in 100 ll PBS on study weeks 0, 3 and 6. Animals in the control group (five mice per group) were administered PBS without VLPs by the same route and according to the same schedule. Blood sample collection. Blood samples were obtained from the tail vein on study weeks 0 (before primary immunization), 2, 4, 6 and 8. All mice were killed on study week 8, and whole blood and spleen tissue were collected.
Serum and splenic lymphocyte preparation Blood samples were centrifuged (Eppendorf, Hamburg, Germany) at 2600 g for 10 min after cooling by storage at 4° for 4–6 hr. The serum was then removed and stored at 20°. Mouse spleen tissues were separated in aseptic conditions, and collected in mouse splenocyte separation medium (Dakewei biotech, Beijng, Biotech, Beijing, China), the structure of the spleen was disrupted using a disposable sterile syringe, and the destroyed issue was filtered using 70-ll cell strainers (BD Falcon, Franklin Lake, NJ). Cell suspensions were centrifuged at 800 g for 30 min (5804R; Eppendorf), to obtain single lymphocytes. After washing once with RPMI-1640 medium (Gibco, Grand Island, NY), The single lymphocytes were resuspended in complete medium consisting of RPMI1640 medium supplemented with 10% fetal calf serum, 1% penicillin and streptomycin, and 1% L-glutamine (Gibco).
Peptide design, synthesis and verification Mouse T-cell epitopes of HBoV VP2 have not yet been reported. So synthetic peptides corresponding to the mouse T-cell epitopes of HBoV VP2 used in ELISPOT assays as specific stimuli were predicted and verified as described previously.23,24 According to the amino acid sequence of the targeted protein, these potential T-cell epitopes were predicted by using computer simulation of the possible spatial structure of polypeptide. Briefly, the whole amino acid sequences of HBoV1 and HBoV2 VP2 were submitted to SYFPEITHI (http://www.syfpeithi.de) and NetMHC 3.2 server (http://www.cbs.dtu.dk/services/ NetMHC). In each genotype, five peptides (two for 15-mers and three for 9-mers) were selected by their scores from high to low in the prediction software and then synthesized by SciLight Peptide (Beijing, China). Peptides were dissolved in RPMI-1640 medium and diluted to the working concentration of 20 lg/ml in complete medium and stored at 20° until further use. ELISPOT assay was performed to identify effective specific peptides. 59
Z.-H. Deng et al. IgG and IgG subtype ELISA The ELISA operation steps were described previously.21 The end-point titres are reported as the highest dilution at which the optical density at 450 nm (OD450) was 21-fold higher than that of the negative control serum.
Specific IgG avidity assay The antibody avidity assay was performed as described previously.25,26 The steps involved were the same as for the IgG and IgG subtype ELISAs, except that after discarding the serum (1 : 200 dilution), 8 M urea (Promega, Madison, WI) was added to wells (200 ll/well) followed by incubation for 5 min at room temperature; this procedure was repeated once to separate the low-activity antibody from the antigen–antibody complex. The avidity index (AI) was calculated as follows: AI ¼ ðOD450 with ureaÞ=ðOD450 without ureaÞ 100% The cut-off for judging the avidity was 50%.
labelled anti-mouse IFN-c antibody (BD Biosciences) was added and incubated for 2 hr at room temperature. After washing three times with PBS-T, horseradish peroxidase-labelled streptavidin was added at a dilution of 1 : 100, and incubated for 1 hr at room temperature. After washing, the spots were developed with a 3-amino9-ethylcarbazole substrate set (BD Biosciences). Spots were counted with a Bioreader (Biosys, Heidelberg, Germany).
Statistical analysis The Mann–Whitney U-test was used to compare the serum IgG titres and analysis of variance (ANOVA) was used to compare the IgG avidities, IgG1/IgG2a titres, and cellular immune responses determined by IFN-c ELISPOT assay in both HBoV1 and HBoV2 immunization groups. Student’s t-test was used to evaluate differences in serum IgG titres, IgG avidities and cellular immune responses determined by IFN-c ELISPOT assay. Statistical analyses were performed using SPSS ver. 17.0 (SPSS, Chicago, IL). In all analyses, P < 005 was taken to indicate statistical significance.
Cross-reaction and cross-reaction avidity assay The assay was based on the ELISAs described above. Sera were collected from mice immunized with HBoV1 or HBoV2 VLPs on study week 8, divided into three equal portions, and diluted 1 : 200 with PBS-T. Two portions were added to 96-well microplates coated with HBoV1 or HBoV2 VLPs, and the third was used for avidity assay and added to microplates coated with HBoV1 or HBoV2 VLPs. The following steps were identical to those of the IgG and IgG subtype ELISAs or specificity IgG avidity assay described above. The cross-reaction rate (CRR) was calculated as follows: CRR ¼ ðOD450 other-VLPs/OD450 self-VLPsÞ 100% The cross-reaction avidity index was calculated as described above.
ELISPOT interferon-c assay Ninety-six-well ELISPOT plates (BD Biosciences, San Diego, CA) were coated at 4° overnight with 05 lg unlabelled mouse interferon-c (IFN-c) antibody (BD Biosciences) and blocked by incubation for 2 hr at room temperature with blocking solution. Splenocytes (5 9 105) were added to each well, and cultivated with HBoV1-specific or HBoV2-specific peptide for 24 hr at 37° in a 5% CO2 atmosphere. Culture medium containing 10 lg/ml concanavalin A (Con A) (Invitrogen) without peptide was used as the control. After aspiration of the cell suspension, wells were washed first with deionized water and then with PBS-T. Then, 02 µg biotin-
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Results Characteristics of HBoV1 and HBoV2 VLPs The molecular weights of high-purity HBoV1 and HBoV2 VLPs were approximately 60 000 (Fig. 1a,b), consistent with previous reports.20,21 On transmission electron microscopy, the VLPs were shown to be approximately 21–24 nm in diameter (Fig. 1c), which is similar to human bocavirus and other parvoviruses reported previously.27,28 The HBoV1 and HBoV2 VLP concentrations determined by BCA protein assay were 1816 and 2964 lg/ml, respectively (data not shown).
A high level of HBoV-specific IgG was induced by HBoV VLPs Administration (i.m. and i.d.) of both HBoV1 (Fig. 2a) and HBoV2 (Fig. 2b) VLPs with alum induced strong HBoV-specific IgG responses in all of the experimental animals. The serum IgG titres of mice at week 8 were subjected to logarithmic transformation and compared (Fig. 2c). There were no differences between the two immunization routes for both HBoV1 and HBoV2 (P > 005), but VLP immunization with alum induced significantly higher IgG titres than VLP immunization alone with both immunogens (P < 001). The IgG titre induced by immunization with HBoV1 VLPs was higher than that induced by immunization with HBoV2 (P < 001).
ª 2013 John Wiley & Sons Ltd, Immunology, 142, 58–66
Immunogenicity of HBoV-1,2 VP2 VLPs in mice (a)
M
HBoV1
HBoV2
(b)
HBoV1
HBoV2 Marker ~175 000 ~130 000 ~95 000 ~70 000
90 000
~62 000
66 000
~51 000 ~42 000
45 000 ~29 000 ~22 000
35 000
~14 000 29 000
~10 500
SDS–PAGE
Figure 1. The purity and molecular weight of human bocavirus type 1 (HBoV1) and HBoV2 VP2 virus-like particles (VLPs) with molecular weight approximately 60 000 used to immunize mice and as antigen in ELISA are shown (15% SDS–PAGE) (a). The specificities of HBoV1 and HBoV2 VP2 VLPs were determined by Western blotting (b). Structures of VLPs were observed by transmission electron microscopy. The diameter was approximately 21–24 nm (c).
Western blot
(c)
100 nm
X97000 HBoV1 VLPs
100 nm
X97000 HBoV2 VLPs
Serum HBoV-specific IgG activity induced by VLP immunization was higher with than without alum adjuvant
ity index than HBoV2-immunized serum (P < 001) (Fig. 4).
The mean avidity indexes of each group are shown in Fig. 3. The IgG avidities induced by immunization with HBoV1 and HBoV2 VLPs through both i.m. and i.d. routes were higher with alum adjuvant than without alum adjuvant. The ANOVA indicated that VLPs injected in conjunction with alum induced higher IgG avidity than VLPs administered alone for both HBoV1 (P < 001) and HBoV2 (P < 001), but there were no differences related to immunization route (i.m. versus i.d.) (P > 005) for both HBoV1 and HBoV2. A Student’s t-test indicated no differences in the IgG avidities induced by HBoV1 and HBoV2 VLPs (P > 005).
Increased cellular immunity was induced by single immunization with VLPs via i.m. injection
Increased cross-reaction avidity was induced by HBoV1 VLPs The cross-reaction rates in HBoV1-immunized serum reacting with HBoV2 VLPs and HBoV2-immunized serum showed no difference between the two subtypes (P > 005). However, the cross-reaction avidities induced by HBoV1 VLPs and HBoV2 VLPs differed; HBoV1 VLP-immunized serum had a higher cross-reaction avidª 2013 John Wiley & Sons Ltd, Immunology, 142, 58–66
The T-cell epitopes of HBoV were predicted (Table 1) and verified by ELISPOT assay Two nine-amino-acid HBoV1-specific epitopes (P3:GYIPIENEL; P5:LYQMPFFLL) and one nine-amino-acid HBoV2-specific epitope (P8:GYIVIHEL) showed high activity and specificity but little cross-reactivity. P3 and P8 were selected as the HBoV1- and HBoV2-specific epitopes, respectively, for the ELISPOT assay. The cellular immune response was then evaluated by ELISPOT assay (Fig. 5). In contrast to the serum IgG antibody response, i.m. and i.d. immunization with VLPs alone induced higher levels of cellular immunity for both HBoV1 and HBoV2 compared with immunization in conjunction with alum adjuvant (i.m., P < 0001; i.d., P < 001). The immunization route, whether i.m. or i.d., did not affect the response induced by HBoV1 VLPs with or without alum (P > 005), or that induced by HBoV2 VLPs with alum adjuvant (P > 005). However, the route of immunization with HBoV2 VLPs without
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Z.-H. Deng et al. HBoV1 VLPs Immunization
(a) Antibody titre (log2)
22 20
VLPs i.m. VLPs+Alum i.m. VLPs i.d. VLPs+Alum i.d.
18 16 14
2
4
6
8
Time (weeks) HBoV2 VLPs Immunization
(b) Antibody titre (log2)
22 20
VLPs i.m. VLPs+Alum i.m. VLPs i.d. VLPs+Alum i.d.
18 16 14
2
4
6
8
Time (weeks) HBoV VLP– induced IgG response
(c)
** 22
**
**
***
IgG titre (log2)
** 20
VLPs i.m. VLPs+Alum i.m. VLPs i.d. VLPs+Alum i.d.
18 16 14
HBoV1 VLPs
HBoV2 VLPs Immunogens
Figure 2. Antibody titres are shown as geometric means. Serum-specific IgG titres increased over time (a, b). Serum IgG titres collected on study week 8 were logarithmically transformed, and the Mann–Whitney U-test was used for comparison between immunization via intradermal (i.d.) and intramuscular (i.m.) routes with or without alum adjuvant. The Student’s t-test was used to compare the IgG titres induced between human bocavirus type 1 (HBoV1) and HBoV2 virus-like particles (VLPs) (c). *P < 005, ** P < 001,*** P < 0001.
HBoV VLP–induced IgG Avidity
Avidity index (%)
100
*
*
*
*
80
VLP i.m. VLP i.d. VLPs+Alum i.m. VLP+Alum i.d.
60 40 20 0
HBoV1
HBoV2 Immunogen
Figure 3. Human bocavirus (HBoV) -specific IgG avidities are shown as the means and standard deviation of avidity index (AI). Sera were collected from mice on study week 8 and diluted 1 : 200 in PBS-T. The AI was calculated as follows[AI = (OD450 with urea)/(OD450 without urea) 9 100%],analysis of variance was used to compare the IgG avidity indexes between immunization via intradermal (i.d.) and intramuscular (i.m.) routes with and without alum adjuvant The Student’s t-test was used to compare the IgG avidity index between HBoV1 and HBoV2 viruslike particles (VLPs). *P < 005.
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Immunogenicity of HBoV-1,2 VP2 VLPs in mice Cross-reaction rate
Cross-reaction avidity index
100
(1 : 290 000) (P < 005), and was also unaffected by the presence or absence of alum adjuvant (data not shown).
**
Rate (%)
80
Discussion
60
40
20
0
HBoV1
HBoV2 Immunogen
Figure 4. Cross-reaction rate was used to describe the proportions of human bocavirus type 2 (HBoV2) and HBoV1 virus-like particle (VLP) combined IgG antibodies in the sera of HBoV1 and HBoV2 VLP-immunized mice, respectively. A cross-reaction index was used to describe the activities of these cross-reactive IgG antibodies. *P < 005, **P < 001 (Student’s t-test). Table 1. List of synthesized peptides Be form
Start position
Amino acid sequence
Number
HBoV1 VP2
260 46 191 130 217 258 120 193 49 187
GLMFNPKVPTRRVQY GSHFSDKYVVTKNTR GYIPIENEL IYNLQIKQI LYQMPFFLL GLMFNPKVPTRRAQY YKRFKPRKMHVKIYN GYIPVIHEL SYFTDSYVI WYLFQYGYI
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10
HBoV2 VP2
The predicted human bocavirus (HBoV) -specific peptides are shown. Five peptides were selected based on higher scores and water solubility for each subtype of HBoV. The start positions of each peptide in the viral protein (VP2) amino acid sequence are shown together with peptide number. Be form means that peptides from which genotype of HBoV.
adjuvant affected (P < 001).
the
IFN-c
response
significantly
The T helper type 1/type 2 immune response induced by VLPs with or without adjuvant The T helper type 1 (Th1)/Th2 type immune responses induced by HBoV VLPs were determined (Fig. 6). The IgG2a and IgG1 antibody titres were logarithmically transformed and compared. HBoV1 VLPs induced a balanced IgG1 and IgG2a response (P > 005), which was not affected by the presence or absence of alum adjuvant. However, HBoV2 VLPs induced a twofold higher IgG1 antibody level (1 : 540 000) compared with that of IgG2a ª 2013 John Wiley & Sons Ltd, Immunology, 142, 58–66
In the present study, HBoV VLPs induced high levels of serum IgG, as reported previously for other VLPs.29 This is because VLPs can efficiently combine with the B-cell receptor, which consists of immunoglobulin molecules, and the interaction between B cells and VLPs induces high levels of neutralizing antibodies.9 Furthermore, the HBoV VLPs induced high titres of IgG antibodies in mice without any adjuvant, which is in accordance with some previous studies.30,31 However, higher IgG titres were induced by immunization with HBoV VLPs with alum adjuvant. Alum may attract antigen-presenting cells to the injection site to recognize and engulf the antigens.32,33 In addition, alum adjuvant was reported to absorb antigens and form an antigen library to continuously stimulate immunocytes.34,35 Therefore, vaccines administered with alum adjuvant can induce higher levels of humoral immunity. These results suggest that HBoV VP2 VLPs could be administered in conjunction with alum adjuvant to improve immunogenicity. High levels of IgG do not necessarily correspond to a high rate of clearance of exogenous virus. However, antibody avidity plays an important role, as high-avidity antibodies bind strongly to exogenous virus. In the present study, i.m. and i.d. immunization with HBoV1 and HBoV2 VLPs with or without alum adjuvant resulted in high IgG avidity. Furthermore, there were no differences in antibody avidity between the two HBoV genotypes, as determined by ANOVA. However, alum adjuvant markedly increased serum IgG avidity, whereas immunization route had no such influence. These results suggest that serum IgG induced by immunization with HBoV VLPs and alum adjuvant more effectively clear exogenous virus and protect against infection, so alum should be used in HBoV VLP vaccination. A previous study indicated serum cross-reactivity between HBoV1 and HBoV2–4,20 which may be a result of the high degree of VP2 gene homology among these viruses. In the present study, the rates of cross-reaction of HBoV1-immunized serum with HBoV2 VLPs and HBoV2-immunized serum with HBoV1 VLPs were 743 96% and 698 152%, respectively. These differences in cross-reactivity were not significant (P > 005). However, the serum avidity cross-reaction rate differed between sera from mice that received immunization with HBoV1 and HBoV2 VLPs. The serum IgG induced by HBoV1 VLPs showed high avidity with HBoV2 VLPs, with an avidity index of 564 120%, which was significantly higher than that of HBoV2immunized serum (326 92%) (P < 001). Cross-neutralizing antibodies can mediate cross-protection.36 These 63
Z.-H. Deng et al. (a)
HBoV1 VLP-induced cellular immune response 250
***
Spots/106 cells
200
***
150 100 50 0 VLPs IM VLPs+Alum VLPs ID VLPs+Alum PBS IM PBS+Alum PBS ID PBS+Alum i.m. i.d. i.m. i.d. Experimental groups
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200 150 ** 100 50 0 VLPs IM VLPs+Alum VLPs ID VLPs+Alum PBS IM PBS+Alum PBS ID PBS+Alum ID IM IM ID Experimental groups
results suggest that HBoV1 VLPs would provide better protection than HBoV2 VLPs when administered at appropriate doses. However, we did not address crossreactivity between HBoV1–2 VLPs and HBoV3 and HBoV4. Characterization of the cellular immune response is an indispensable part of the evaluation of a candidate vaccine. Measurement of the IFN-c level is often used to evaluate the cellular immune response. In the present study, IFN-c production was induced in the splenocytes of mice immunized with HBoV1 and HBoV2 VLPs. These data confirm that both HBoV1 and HBoV2 VLPs can activate T cells and stimulate cellular immunity. Statistical analysis indicated that alum adjuvant had the opposite effect on the cellular immune response. This is contrary to what it does in the humoral immune response. That is, the cellular immune response, as monitored by IFN-c ELISPOT assay, was induced more strongly by immunization with VLPs alone compared with by immunization with VLPs plus alum adjuvant. It is known that aluminium adjuvants can advantageously regulate lymphocyte differentiation into helper T lymphocytes and enhance the humoral immune response, but weaken cytotoxic T lymphocyte differentiation and prolif64
Figure 5. Production of interferon-c (IFN-c) as a cellular immunity-associated cytokine was determined by ELISPOT assay. The effective human bocavirus (HBoV) -specific peptides were selected from Table 1, HBoV1-specific P3 (GYIPIENEL) and HBoV1-specific P8 (GYIPVIHEL) were used as specificity stimuli. The ELISPOT results were converted into spots/106 cells. The results for each group are shown as the means and standard deviation (a, b). Analysis of variance was used to compare the spots between immunization via intradermal (i.d.) and intramuscular (i.m.) routes with or without alum adjuvant. *P < 005, **P < 001, ***P < 0001.
eration.37 This seemed to be because the antigen library established by injection with alum adjuvant prolongs the stimulation, but also reduces the intensity of stimulus at the same time, and the presence of alum did not improve the cellular immune response.32,38 The cellular immune responses of the two genotypes were not compared because of the different specific stimulus peptides. However, there have been no previous studies of the T-cell epitopes of HBoV in mice. The sequences of stimulus peptides used in the present study were predicted using a bioinformatics method described previously.23,24 Our results confirmed that HBoV VLPs induced a specific cellular immune response, but do not reflect the entire cellular immune response in vivo, and further studies that compare all possible T-cell epitopes are required. Detection of IgG1 and IgG2a is usually performed to determine whether vaccine-elicited immunity tends toward the Th1 or Th2 type. In mice, the Th1-type immune response produces IgG2a subtype antibodies, whereas the Th2-type immune response produces IgG1 subtype antibodies. However, the immune response in humans is the reverse; i.e. IgG1 reflects a Th1-type immune response, while IgG2a reflects a Th2-type immune response.29,39,40 Some previous reports indicate ª 2013 John Wiley & Sons Ltd, Immunology, 142, 58–66
Immunogenicity of HBoV-1,2 VP2 VLPs in mice (a)
HBoV1 VLP immunization 3 VLP-induced IgG1 VLP-induced IgG2a
2·5
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OD 450
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PBS control group mice IgG1 PBS control group mice IgG2a
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VLP-induced IgG1 VLP-induced IgG2a VLP+Alum-induced IgG1 VLP+Alum-induced IgG2a PBS control group mice IgG1 PBS control group mice IgG2a PBS+Alum control group mice IgG1 PBS+Alum control group mice IgG2a
2·5
OD 450
2 1·5 1 0·5
that VLPs usually induce a balanced Th1/Th2 immune response;29 this was indeed found in the present study. The end-point titres of IgG1/IgG2a in serum induced by HBoV VLP immunization were determined by ELISA. The results indicated that HBoV1 VLPs induced a balance of Th1/Th2 immune response, reflected by high but not significantly different serum IgG2a/IgG1 antibody titres. However, the immune response induced by HBoV2 VLPs was different from that induced by HBoV1 VLPs. That is, the serum IgG1 titre induced by HBoV2 VLPs was higher than the serum IgG2a antibody titre. This suggests that HBoV2 VLPs induce a stronger IgG1-subtype immune response than IgG2a-subtype immune response, which is indicative of a stronger Th2-type than Th1-type immune response. In conclusion, our data show that HBoV VP2 VLPs have good immunogenicity and induce strong humoral and cellular immune responses in mice. Therefore, HBoV VP2 VLPs represent good candidate proteins for HBoV vaccine. It should be noted that we did not determine the protective efficacy of this VLP and did not perform a neutralization test without live virus and HBoV-sensitive animals or cells, which are essential in a neutralization test. Recently, successful production of the HBoV1 virons in vitro through transfection of a plasmid-like HBoV into human embryonic kidney 293 cells has been reported and ª 2013 John Wiley & Sons Ltd, Immunology, 142, 58–66
·4 76 ·8 32
2
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81
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Figure 6. Sera were collected from mice on study week 8. The results of IgG1 and IgG2a subtype antibodies are shown as means of OD450 from ELISA. Serum dilutions were doubled starting from 1 : 100.
Serum dilution (×1000)
these virions are able to infect human airway epithelial cells,41 which will promote HBoV vaccine development.
Acknowledgements We are grateful to Baoying Huang and Jingdong Song for technical assistance during the study. This work was partly supported by a research conditions innovation project grant (2012TT2004) from the Science and Technology Agency of Hunan Province, China.
Disclosures None of the authors has a conflict of interest.
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