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C3d DNA followed by a recombinant pseudorabies virus boost enhanced protective immunity against H3N2 swine influenza virus in mice
Research in Veterinary Science 88 (2010) 345–351
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Prime–boost immunization with HA/C3d DNA followed by a recombinant pseudorabies virus boost enhanced protective immunity against H3N2 swine influenza virus in mice Guo-Xin Li a,b, Yan-Jun Zhou a, Hai Yu a, Zhi-Jun Tian b, Li-Ping Yan a, Qiang Zhang c, Shou-Ping Hu b, Guang-Zhi Tong a,b,* a
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518 Ziyue Road, Minhang District, Shanghai 200241, China Division of Swine Infectious Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 427 Maduan Street, Harbin 150001, Heilongjiang, China c Technical Center for Animals, Plants and Foods Inspection and Quarantine Shanghai Entry-Exit Inspection and Quarantine Bureau PRC, No. 1208 Minsheng Road, Pudong New Area, Shanghai 200135, China b
a r t i c l e
i n f o
Article history: Accepted 15 September 2009
Keywords: Swine influenza virus DNA vaccines C3d Pseudorabies virus Prime–boost
a b s t r a c t DNA and recombinant virus vaccines against swine influenza virus (SIV) have been pursued with promising results, but induce poor immunogenicity. This study evaluated the effects of a vaccine regimen in mice including priming with three DNA vaccines expressing soluble HA (sHA), complete HA (tmHA), or sHA fused with three copies murine C3d (sHA–mC3d3) and boosting with recombinant pseudorabies virus expressing HA (rPRV–HA). Immune responses were monitored by ELISA, HI assays, and virus neutralization. Protective efficacy was evaluated by virus isolation from lungs, distribution in tissues, and pathology following challenge with H3N2 SIV. Priming with sHA–mC3d3 and boosting with rPRV–HA induced higher levels of HA-specific antibodies and yielded the most effective protection. This finding implied that priming with a DNA vaccine expressing C3d fused with antigen and boosting with a recombinant vector vaccine is an effective way to induce protective humoral immunity and prevent some infectious diseases. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Swine influenza virus (SIV) causes severe respiratory disease characterized by an acute explosive outbreak of symptoms such as coughing, high fever, nasal discharge, anorexia, and weight loss (Olsen, 2002). Under field conditions, coinfection of SIV with other pathogens could result in a significant negative economic impact for the swine industry (Choi et al., 2003). Beyond veterinary implications, influenza virus infection in pigs also poses an important public health concern. Swine are referred to as a ‘‘mixing vessel” because of their susceptibility to both human and avian influenza viruses (Ito et al., 1998; Castrucci et al., 1993). Reassortment of avian and mammalian influenza viruses in swine may produce new viruses, some of which may have the potential to transmit to humans (Webster et al., 1992; Karasin et al., 2000). Therefore, it is important to develop effective strategies to control swine influenza to prevent virus replication in the swine ‘‘mixing vessel”,
* Corresponding author. Address: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518 Ziyue Road, Minhang District, Shanghai 200241, China. Tel.: +86 21 54113138; fax: +86 21 54081818. E-mail address: [email protected] (G.-Z. Tong). 0034-5288/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2009.09.005
thus decreasing the possibility of creating potentially pandemic influenza virus reassortments. Inactivated vaccines are commercially available, but new vaccines that are capable of inducing virus-specific neutralizing antibody plus cell-mediated immunity will provide superior protection against acute influenza diseases (Wesley et al., 2004). In recent years, as an alternative to conventional swine influenza vaccines, DNA vaccines and recombinant virus vaccines have been pursued with some positive and promising results (Macklin et al., 1998; Wesley et al., 2004; Endo et al., 1991; Larsen et al., 2001; Tang et al., 2002). Our recent study showed that three repeated inoculations of DNA vaccines expressing different forms of hemagglutinin (HA) elicited specific immune responses and protected mice from homologous SIV challenge. Although the results were promising, the requirement for three injections makes the DNA vaccines more theoretical than practical for use in swine (Li et al., 2009). In another study, we constructed a recombinant pseudorabies virus expressing HA (rPRV–HA) which protected mice from homologous SIV challenge, although only weak antibody responses were induced (Tian et al., 2006). Recombinant virus vaccines are not suitable for booster immunization when priming with vaccines produced in the same vector
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because the immunity induced by prior immunization may neutralize or inactivate the vector and interfere with antigen presentation. The main disadvantage of DNA vaccines is their poor immunogenicity, especially in large animals. Large quantities of DNA are required to induce only modest immunogenicity (Graham et al., 2006). To circumvent the above problems, we explored a combination vaccine strategy. The immune response was primed with an antigen delivered by one vector, and then boosted using the same antigen delivered by an immunologically distinct vector to augment immune response and protection (Newman, 2002). Several studies have shown that a prime–boost immunization regimen with a DNA plasmid and recombinant virus vaccine, both expressing the same antigen, can induce a strong immune response, including cell-mediated immunity (Schneider et al., 1998; Dégano et al., 1999; Amara et al., 2001). Furthermore, a regimen including a DNA prime and inactivated influenza vaccine boost induced stronger immune responses than did the prime– boost using inactivated vaccine or DNA vaccines alone (Larsen et al., 2001; Wang et al., 2008). However, it is unknown whether a DNA vaccine expressing soluble HA, complete HA, or a fusion of HA with a molecular adjuvant is more effective in heterologous prime–boost immunization regimens. The purpose of the present study was to determine if a vaccine strategy including priming with HA-expressing DNA and boosting with rPRV–HA could enhance immune responses and protection efficiency against homologous SIV challenge. Our previous study showed that a DNA vaccine expressing a fusion of soluble HA with three copies of murine C3d (sHA–mC3d3) induced a stronger immune response than a DNA vaccine expressing soluble HA (sHA) or complete HA (tmHA) (Li et al., 2009). This study determined that priming with sHA–mC3d3 was more effective than priming with either sHA or tmHA when boosting with rPRV–HA. 2. Materials and methods 2.1. Viruses and cell cultures SIV strain A/Swine/Heilongjiang/74/2000 (H3N2) (SwHLJ74) was provided by Dr. Li at Harbin Veterinary Research Institute, Harbin, China. PRV Bartha-K61 strain and recombinant pseudorabies virus expressed HA (rPRV–HA) were propagated and titrated in PK-15 or Vero cells as previously described (Tian et al., 2006). All cells were grown and maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U/ml ampicillin, and 100 lg/ml streptomycin. 2.2. HA–DNA vaccines The HA–DNA vaccines encoding complete HA (tmHA), soluble HA (sHA), or a soluble fused form of HA (sHA–mC3d3) were previously constructed from the H3N2 subtype of SIV (Li et al., 2009). tmHA expresses full-length wild-type HA. sHA was generated by deleting the transmembrane and cytoplasmic domains of HA and replacing the tissue plasminogen activator (tPA) leader sequence with a signal peptide. sHA–mC3d3 was generated by inserting three copies of murine C3d downstream of sHA (Fig. 1). All plasmids were amplified in Escherichia coli strain DH5a and were purified using anion-exchange resin columns (Qiagen). 2.3. Immunization of mice Eight-week-old BALB/c female mice were obtained from the Laboratory Animal Center of Harbin Veterinary Research Institute for immunogenicity studies. Animal maintenance and experimental protocols were approved by the Animal Experiment Ethics
Natural leader Transmembran A
tmHA
HA1
HA2
B
sHA
HA1
HA2
tPA leader C
sHA/mC3d3
HA1
Linker HA2
mC3d
mC3d
mC3d
Fig. 1. Schematic representation of DNA vaccine constructs. (A) Structure of the wild-type, transmembrane form of HA. (B) Structure of extracellular part of HA, linked with a signal sequence of tPA encoding a secreted HA (sHA). (C) Structure of sHA linked with three copies of murine C3d (sHA–mC3d3). Two repeats of four glycines and one serine {(G4S)2} as the linkers were inserted at the junctures of HA and mC3d and between each mC3d repeat.
Committee of the authors’ institute. The mice (18 mice per group) received two immunizations at weeks 0 and 4 with different combination immunizations (Table 1). Animals were injected intramuscularly (i.m.) with 100 lg plasmid DNA or inoculated intranasally (i.n.) with 105 PFU of rPRV–HA as indicated in Table 1. Sera were collected at 0, 2, 4, 6, and 8 weeks after the primary immunization to detect specific antibodies. At week 8 after primary immunization, mice were challenged i.n. with 105 TCID50 of SwHLJ74 (H3N2). 2.4. Serological assay An endpoint ELISA was performed to assess the titers of HA-specific antibody. Purified influenza virus was used to coat plates as described previously (Chen et al., 2007). Endpoint dilution titers from immunized mice that were twofold higher than sera from mice in control groups were considered positive. The hemagglutinin inhibition (HI) assay was conducted as previously described (Robinson et al., 1997) using four hemagglutination units of SIV virus. Neutralization assays were conducted as previously reported (Torres et al., 2000). Neutralization titers were reported as the highest dilution giving complete inhibition of replication of MDCK cells given a TCID50 of 100 in 50 ll DMEM medium. The presence of replicating virus in a well was scored by hemagglutination. 2.5. Lymphocyte proliferative responses Lymphocyte proliferative responses using mouse splenocytes were detected as previously described (Li et al., 2009). Briefly, four weeks after boost immunization splenocytes were isolated from immunized mice, suspended in RPMI 1640, and seeded into 96well plates at 4 105 cells per well (200 ll). The cultures were stimulated with either Con A (positive control), 20 ll of inactivated and purified swine influenza virus as the specific antigen, or nothing (negative control). The proliferative response was measured by adding 20 ll WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) to each well with a further 5 h of incubation. The stimulation index was calculated as the ratio of the average OD values in wells containing antigen-stimulated cells to the average OD of wells containing only cells with medium. 2.6. IL-4 and IFN-c release assay At week 4 after boost immunization, the spleens from three mice from each group were harvested. Mouse splenocytes were prepared as described (Bounous et al., 1992) and incubated at 37 °C in 96-well plates at a concentration of 2 105 cells per well in the presence of 20 ll inactivated and purified SIV. After 72 h incubation, supernatants were harvested and the presence of IL-4
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G.-X. Li et al. / Research in Veterinary Science 88 (2010) 345–351 Table 1 Vaccination schedule and challenge of mice. Immunization and challenge
Groups sHA
tmHA
sHA/mC3d3
sHA + rPRV–HA
tmHA + rPRV–HA
sHA/mC3d3 + rPRV–HA
rPRV–HA
Control
Prime (week 0) Boost (week 4)
sHA sHA
tmHA tmHA
sHA/mC3d3 sHA/mC3d3
sHA rPRV–HA
tmHA rPRV–HA
sHA/mC3d3 rPRV–HA
Vector rPRV–HA
Vector Vector
Challenge (week 8)
Challenged with SwHLJ74 (H3N2)
and IFN-c was tested with mouse IL-4 and IFN-c ELISA kits (Biosource, USA) according to the manufacturer’s instructions. The concentrations of mouse IL-4 and IFN-c in the samples were determined from the standard curves.
3. Results
2.7. Virus detection and isolation
Levels of anti-HA IgG responses in immunized mice were measured by ELISA. At week 2 after the first inoculation, the antibody titer exceeded 1:200 in all groups immunized with HA–DNA, while the antibody titer of control group mice was undetectable. At week 4 after boost immunization, the anti-HA IgG titer elicited by sHA– mC3d3 in the DNA prime–boost groups was significantly higher than the titers elicited by sHA and tmHA (P < 0.01, Fig. 2). Furthermore, the DNA prime–rPRV–HA boost elicited higher anti-HA IgG titers compared with the corresponding DNA prime–boost groups or the rPRV–HA only group (Fig. 2). The antibody titers elicited by sHA prime–rPRV–HA boost and tmHA prime–rPRV–HA boost were similar. Interestingly, sHA–mC3d3 prime–rPRV–HA boost elicited the highest anti-HA IgG titers among these groups. The differences in the anti-HA IgG levels between sHA–mC3d3 prime– rPRV–HA boost and any other DNA prime–rPRV–HA boost was statistically significant (P < 0.01, Fig. 2).
Five mice in each group were euthanized 3, 7, and 10 days postchallenge (DPC). At necropsy, lung, spleen, kidney, and cerebrum samples were collected and analyzed by immunofluorescence assay (IFA) (Yoon et al., 1992). Nine- to eleven-day-old embryonated chicken eggs were inoculated allantoically with 0.2 ml of the lung samples and incubated at 37 °C for 72 h. The allantoic fluids were then harvested and tested for hemagglutinin activity. Positive samples were further characterized by the HI test. 2.8. Histopathology Thin sections of formalin-fixed, paraffin-embedded tissues were taken from the lungs, spleen, kidney, and cerebrum and stained using hematoxylin–phloxin–safran (HPS) as described previously (Dea et al., 1991). 2.9. Statistical analysis All data were presented as the mean ± standard error (SE). The presence of statistical differences among groups was determined by one-way ANOVA; the method of least significant difference (LSD or Dunnett T 3 test) was used to compare the effects among groups with the aid of SPSS 13.0 for windows. Differences were deemed statistically significant when the P-value was <0.05.
sHA
3.2. Hemagglutinin–inhibition (HI) and virus neutralization (VN) titers The levels of functional antibodies in sera from immunized mice were measured using hemagglutination inhibition and microneutralization assays (Figs. 3 and 4). At week 4 after boost immunization, HI and VN titers elicited by sHA–mC3d3 in the DNA prime–
tmHA+rPRV-HA
tmHA
sHA/mC3d3+rPRV-HA
sHA/mC3d3
rPRV-HA
sHA+rPRV-HA
Control
sHA
tmHA+rPRV-HA
tmHA
sHA/mC3d3+rPRV-HA
sHA/mC3d3
rPRV-HA
sHA+rPRV-HA
Control
250 d 200
HI antibody titers
Endpoint dilution titers
100000
3.1. Antibody responses to prime–boost immunizations detected by ELISA
10000
1000
c 150
bc
bc
b 100 b
b
50 a
< 100 0
2 4 6 Weeks after primary immunization
8
Fig. 2. Anti-HA IgG levels in mice after vaccination. Mice were primed at week 0 and boosted at week 4. Sera were collected from five mice per group for determination of specific IgG levels by ELISA.
0 Fig. 3. The hemagglutination inhibition (HI) antibody responses in sera immunized with DNA vaccines, rPRV–HA, or the combination of both. The HI antibody titers are shown as the geometric means for each group (five mice) with standard errors. Means not sharing a common letter are significantly different at P < 0.05.
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sHA
tmHA+rPRV-HA
tmHA
sHA/mC3d3+rPRV-HA
sHA/mC3d3
rPRV-HA
sHA+rPRV-HA
Control
sHA tmHA sHA/mC3d3 sHA+rPRV-HA
tmHA+rPRV-HA sHA/mC3d3+rPRV-HA rPRV-HA Control
2.6
6000 2.4
c 5000
NAb titers
4000
Stimulation index
2.2
b
3000 2000 1000
2 1.8 1.6 1.4
a a
a
a 1.2
a a
0 Fig. 4. The neutralizing antibody (NAb) responses in sera immunized with DNA vaccines, rPRV–HA, or the combination of both. The NAb titers against H3N2 virus infection of MDCK cells are shown as the geometric means from each group (five mice) with standard errors. Means not sharing a common letter are significantly different at P < 0.05.
boost groups were significantly higher than the titers raised by sHA and tmHA (P < 0.05; Figs. 3 and 4). The results indicated that C3d was able to stimulate specific B cells to increase the production of antibodies to SIV. Furthermore, the DNA prime–rPRV–HA boost elicited higher levels of HI and VN when compared to the corresponding DNA prime–boost regimens or rPRV–HA alone. Particularly, the sHA–mC3d3 prime–rPRV–HA boost elicited the highest HI and VN titers among these groups.
1 Fig. 5. Lymphocyte proliferative responses at week 4 after boost immunization. The splenocytes were isolated and after 72 h of stimulation with SIV, WST-8 was added. The OD value determined after further 5 h incubation. Data represent the means plus standard errors.
duced by sHA or tmHA. In contrast, tmHA induced a numerically higher level of IFN-c compared with sHA and sHA–mC3d3. The results indicated that C3d fusion elicited a Th2-biased immune response by inducing IL-4 production while the full-length form of HA (tmHA) tended to produce a Th1-biased immune response. Furthermore, the DNA prime–rPRV–HA boost elicited a higher level of IL-4 and IFN-c compared with the corresponding DNA prime– boost groups or the rPRV–HA group (Fig. 6). Interestingly, the sHA–mC3d3 prime–rPRV–HA boost induced the highest level of IL-4 while the tmHA prime–rPRV–HA boost induced the highest level of IFN-c (Fig. 6).
3.3. Lymphocyte proliferation assay
3.5. Virus distribution after challenge
Lymphocyte proliferative responses for three mice from each group were analyzed at week 4 after boost immunization. Proliferation responses in all seven groups immunized with HA–DNA, rPRV–HA, or the combination of both were higher than those of the controls (Fig. 5). For the DNA prime–boost groups, the stimulation index of mice injected with tmHA was higher than that of mice injected with sHA or sHA–mC3d3 (Fig. 5). Furthermore, the DNA prime–rPRV–HA boost elicited increased cell proliferation compared with the corresponding DNA prime–boost groups or the rPRV–HA only group. The proliferation responses elicited by the sHA prime–rPRV–HA boost and the sHA–mC3d3 prime–rPRV–HA boost were similar. However, the tmHA prime–rPRV–HA boost elicited the highest level of proliferative responses among these groups (Fig. 5).
The distribution of SIV antigens in different tissues of immunized animals at necropsy was examined by IFA. Many SIV antigens were detected in the lungs of control animals from 3 to 10 DPC. Less virus antigen was detected in the lungs of DNA or rPRV–HA immunized mice 3 DPC and no virus antigens were detected 7 or 10 DPC. Moreover, fewer virus antigens were detected in the lungs of mice with DNA prime–rPRV–HA boost compared with the mice with DNA prime–boost or the rPRV–HA only 3 DPC (Table 2). SIV antigens could be detected in the spleens of control mice 3 DPC, but not in those of the DNA or rPRV–HA inoculated mice (Table 2).
3.4. Cytokine production by splenocytes from vaccinated mice At week 4 after boost immunization, the spleens from three mice from each group were harvested. Splenocytes from each group were isolated, restimulated with inactivated swine influenza virus, and assessed for the presence of IFN-c, an indication of a Th1 response, and IL-4, an indication of a Th2 response. Mice immunized with HA–DNA, rPRV–HA, or the combination of both showed a rise in IL-4 and IFN-c (Fig. 6). For the DNA prime-boost groups, the level of IL-4 induced by sHA–mC3d3 was higher than that in-
3.6. Isolation of challenge virus from lungs In general, virus was isolated from fewer mice in the DNA prime–rPRV–HA boost groups than that of mice in the corresponding DNA prime–boost groups. At 3 DPC, virus was isolated from all control mice, but no virus was isolated from mice from the sHA– mC3d3 prime–rPRV–HA boost group. The percentage of mice from which virus was isolated dropped in the sHA group (2/5) and tmHA group 2 (1/5) at day 7 post-challenge, and no virus was isolated from mice from the other immunized groups. By 10 DPC, virus could be isolated from only 3/5 control mice, and no virus was isolated from the DNA or rPRV–HA immunized groups (Table 3). These results indicated that immunization with DNA, rPRV–HA, or a combination of the two strategies was able to inhibit replica-
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sHA tmHA sHA/mC3d3 sHA+rPRV-HA
A
tmHA+rPRV-HA sHA/mC3d3+rPRV-HA rPRV-HA Control
Table 3 Isolation of swine influenza virus from the lungs of immunized mice following challenge with SwHLJ74.
1400 1200 1000
pg/ml
800
a
Groups
3 DPC
7 DPC
10 DPC
sHA tmHA sHA/mC3d3 sHA + rPRV–HA tmHA + rPRV–HA sHA/mC3d3 + rPRV–HA rPRV–HA Control
4/5a 4/5 1/5 2/5 1/5 0/5 2/5 5/5
2/5 1/5 0/5 0/5 0/5 0/5 0/5 5/5
0/5 0/5 0/5 0/5 0/5 0/5 0/5 3/5
Number of mice with positive results/total mice.
600 tion of homologous SIV to a great extent and sHA–mC3d3 prime– rPRV–HA boost was the most effective at inhibiting virus replication among these immunization regimens.
400 200
3.7. Pathological findings after challenge
0
B
IL-4 sHA tmHA sHA/mC3d3 sHA+rPRV-HA
tmHA+rPRV-HA sHA/mC3d3+rPRV-HA rPRV-HA Control
1600 1400 1200
pg/ml
1000 800 600 400
Microscopic lesions were observed in most of the control group mice following SIV challenge. Observed lesions included thickened alveolar septa and infiltration of lymphocytes leading to mild to severe interstitial pneumonia (Fig. 7), collapse of follicles, depletion of germinal centers, blurred white medulla, hyperplastic red medulla in spleens (Fig. 7), glial cell nodule formation and liquefaction in the cerebra, slight stroma hyperplasy, and lymphocyte infiltration in kidneys (Table 4). In most of the immunized groups, a few mice showed mild interstitial pneumonia, characterized by slightly thickened alveolar septa, while no pathological lesions were found in the kidneys or cerebra (Table 4). Furthermore, most immunized mice had follicle hyperplasia and germinal center enlargement in spleens, possibly indicative of an immune response to the vaccination. Interestingly, in the sHA–mC3d3 prime–rPRV– HA boost group, none of mice showed microscopic lesions in the lungs, spleens, kidneys, or cerebrum (Table 4). These results indicated that DNA prime–rPRV–HA boost, especially the sHA– mC3d3 prime–rPRV–HA boost, yielded more effective protection than did DNA prime–boost.
200 0
4. Discussion
IFN-gamma
Fig. 6. Levels of IL-4 (A) and IFN-c (B) from splenocytes in mice immunized with DNA vaccines, rPRV–HA, or the combination of both. At week 8 after primary immunization, splenocytes from each mouse group were harvested and were cultured at 2 105 cells per well in 96-well plates and stimulated with inactivated swine influenza virus. IFN-c and IL-4 production in the supernatant was analyzed by ELISA. Data represent the means plus standard errors.
DNA immunization is a promising vaccine strategy against infectious diseases. However, poor immunogenicity has been an obstacle for the clinical application of DNA vaccines. In order to circumvent this problem, various strategies, such as adjuvant augmentation and delivery system optimization, have been considered (Richard and Anderson, 2007). In our previous study, the C3d molecular adjuvant fused with SIV hemagglutinin (sHA–
Table 2 The distribution of swine influenza virus in different organs of immunized mice on different days following challenge with SwHLJ74. Groups
sHA tmHA sHA/mC3d3 sHA + rPRV–HA tmHA + rPRV–HA sHA/mC3d3 + rPRV–HA rPRV–HA Control
3 DPC
7 DPC
10 DPC
Lungs
Spleens
Lungs
Spleens
Lungs
Spleens
+++ +++ +++ ++ ++ ++ +++ ++++
– – – – – – – +
– – – – – – – ++++
– – – – – – – –
– – – – – – – ++
– – – – – – – –
Tissues were collected from all mice tested, with two frozen sections for each tissue, and examined by indirect immunofluorescence antibody test: (–) no stained cells in a whole section; (+) 1–2 positive-stained cells in a section; (++) 1–2 stained cells in one view field; (+++) 2–10 stained cells in one view field; (++++) >10 stained cells in one view field.
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Fig. 7. Histological lesions of mice in sHA–mC3d3 prime–rPRV–HA boost (A–B) and control groups (C–D) 7 days post-challenge with SwHLJ74. (A) Spongiform aspect of the lungs, showing well-delineated, thin alveolar septa with the normal aspect of a small blood vessel. (B) Lymph follicle hyperplasia in spleen. (C) Interstitial pneumonia with alveolar septa thickened by lymphocyte infiltration. (D) Collapsed follicle in spleen. H&E staining. Magnification 40.
Table 4 Gross lesions of mice at necropsy after challenge with SwHLJ74. Groups
Lungs
Kidneys
Cerebrum
Spleen
sHA tmHA sHA/mC3d3 sHA + rPRV–HA tmHA + rPRV–HA sHA/mC3d3 + rPRV–HA rPRV–HA Control
7/15a 6/15 4/15 4/15 3/15 0/15 4/15 10/15
0/15 0/15 0/15 0/15 0/15 0/15 0/15 7/15
0/15 0/15 0/15 0/15 0/15 0/15 0/15 5/15
0/15 0/15 0/15 0/15 0/15 0/15 0/15 9/15
Tissue lesions were scored as: lung, interstitial pneumonia with alveolar septa thickened by infiltration of lymphocytes and macrophages surrounding bronchiole; spleen, collapsed follicles; cerebrum, glial cell nodule formation or liquefaction in cerebrum; kidney, slight stroma hyperplasia and infiltration of lymphocytes. a Number of mice showing pathological lesions/total mice.
mC3d3) enhanced protective immunity against SIV in mice (Li et al., 2009). Large doses of DNA (three inoculations, total 300 lg) were given to induce strong immune responses in that experiment. However, multiple doses may make this vaccine strategy difficult to implement in swine in practice. Thus, in the current study, we investigated the effects of fewer doses (two inoculations, total 200 lg) of DNA inoculation. The results showed that two inoculations of sHA–mC3d3 induced HA-specific immune responses and protected mice from homologous SIV challenge. As such, in future studies, it is important to assess the effects of the DNA vaccine in the context of further decreasing doses or times of injection. In the current study, we compared the levels of immune response and protection efficiency between homologous (DNA + DNA or rPRV–HA alone) and heterologous (DNA + rPRV–HA) prime–boost vaccination strategies. DNA has been considered an
effective priming vector because of the long lasting expression period and the induction of cellular and/or humoral immunity (Ramshaw and Ramsay, 2000). Several recombinant vectors have been developed with the aim of inducing the prolonged expression of high levels of viral antigens, such as adenovirus, pseudorabies virus, fowlpox virus, semliki forest virus, and vesicular stomatitis virus (Richard and Anderson, 2007). However, immune responses elicited by these recombinant virus vaccines targeted not only the foreign antigens but also the vector itself, and pre-existing immunity to the vector has been shown to dramatically decrease vaccination efficiency (Sharpe et al., 2001). DNA prime and recombinant viral vector boost immunization regimens appear to be very effective at inducing high levels of immune responses (Schneider et al., 1998; Dégano et al., 1999; Meseda et al., 2002; Dory et al., 2006). In this study, heterologous DNA prime and rPRV–HA boost in mice elicited higher levels of protective antibodies and yielded more effective protection compared to homologous DNA prime– boost immunization regimens or the single rPRV–HA immunization. However, studies with heterologous prime–boost vaccine regimens in the past have provided conflicting results. Breathnach’s research into influenza vaccines showed that recombinant modified vaccinia virus Ankara (rMVA) induced protective immunity in horses independent of a DNA prime (Breathnach et al., 2006), while a heterologous prime–boost immunization regimen with DNA and rMVA was effective in mice (Dégano et al., 1999). The differences observed in these studies may be partly attributed to differences in the animal models. Therefore, it is important to evaluate the effects of DNA prime–rPRV–HA boost immunization regimens and single rPRV–HA immunization in swine in future studies. Although a previous study indicated that the DNA prime may be more effective than the inactivated influenza vaccine in priming
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the immune system (Wang et al., 2008), in this study sHA–mC3d3 prime–rPRV–HA boost induced higher levels of HA-specific antibodies compared to either sHA or tmHA prime and rPRV–HA boost. However, it is possible that DNA immunization is more effective in eliciting better and potentially longer lasting HA-specific B cell memory (Wang et al., 2008). Low-dose antigen delivery has been shown to be more effective in eliciting better antibody responses and B cell memory (González-Fernández and Milstein, 1998; Bot et al., 1997). Therefore, DNA vaccines may be more suitable as priming vectors than boosting vectors. Interestingly, our results suggested that fusion of C3d with HA may be more effective as a priming immunogen compared to the non-fusion form HA. This result was supported in a previous study that found fusion of C3d with HA may induce affinity maturation of the antibody response and elicit memory B cells more effectively than non-C3d fused forms of HA (Ross et al., 2000). Therefore, the fusion of C3d with antigen priming followed by boosting with other vaccines, such as recombinant vector vaccines or inactivated vaccines, may be an attractive solution to inducing humoral protective humoral immunity to some infectious diseases. In future studies there are a number of areas which should be explored, including the mucosal immune response induced by these vaccination regimens and evaluation of the effects of priming with various doses of DNA. Furthermore, since H1N1 and H3N2 are the dominant subtypes causing swine influenza worldwide and can be found co-circulating in the same herd with some frequency, it is important to determine whether such prime–boost regimens are able to provide protective immunity against heterologous strains of SIV. Acknowledgements The study was supported by Grants from the National Basic Research Program (973) of China (No. 2005CB523200), the National High-Tech Research and Development Program (863 Program) of China (No. 2006AA10A204) and the National Scientific Supporting Program of China (No. 2006BAD06A04/03/01). References Amara, R.R., Villinger, F., Altman, J.D., Lydy, S.L., O’Neil, S.P., Staprans, S.I., et al., 2001. Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science 292 (5514), 69–74. Bot, A., Antohi, S., Bona, C., 1997. Immune response of neonates elicited by somatic transgene vaccination with naked DNA. Front. Biosci. 2, d173–d188. Bounous, D.I., Campagnoli, R.P., Brown, J., 1992. Comparison of MTT colorimetric assay and tritiated thymidine uptake for lymphocyte proliferation assays using chicken splenocytes. Avian. Dis. 4, 1022–1027. Breathnach, C.C., Clark, H.J., Clark, R.C., Olsen, C.W., Townsend, H.G., Lunn, D.P., 2006. Immunization with recombinant modified vaccinia Ankara (rMVA) constructs encoding the HA or NP gene protects ponies from equine influenza virus challenge. Vaccine 24 (8), 1180–1190. Castrucci, M.R., Donatelli, I., Sidoli, L., Barigazzi, G., Kawaoka, Y., Webster, R.G., 1993. Genetic reassortment between avian and human influenza A viruses in Italian pigs. Virology 193 (1), 503–506. Chen, Y., Xin, X.G., Yang, H.L., Li, T., Li, H.Y., Qiao, C.L., et al., 2007. Establishment of an indirect ELISA for detection antibodies against swine influenza virus. Prog. Vet. Med. 4, 308–311 (in Chinese). Choi, Y.K., Goyal, S.M., Joo, H.S., 2003. Retrospective analysis of etiologic agents associated with respiratory diseases in pigs. Can. Vet. J. 44 (9), 735–737. Dea, S., Bilodeau, R., Sauvageau, R., Martineau, G.P., 1991. Outbreaks in Quebec pig farms of respiratory and reproductive problems associated with encephalomyocarditis virus. J. Vet. Diagn. Invest. 3 (4), 275–282. Dégano, P., Schneider, J., Hannan, C.M., Gilbert, S.C., Hill, A.V., 1999. Gene gun intradermal DNA immunization followed by boosting with modified vaccinia virus Ankara: enhanced CD8+ T cell immunogenicity and protective efficacy in the influenza and malaria models. Vaccine 18 (7–8), 623–632. Dory, D., Fischer, T., Béven, V., Cariolet, R., Rziha, H.J., Jestin, A., 2006. Prime–boost immunization using DNA vaccine and recombinant Orf virus protects pigs against Pseudorabies virus (Herpes suid 1). Vaccine 24 (37–39), 6256–6263.
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Karasin, A.I., Schutten, M.M., Cooper, L.A., Smith, C.B., Subbarao, K., Anderson, G.A., et al., 2000. Genetic characterization of H3N2 influenza viruses isolated from pigs in North America, 1977–1999: evidence for wholly human and reassortant virus genotypes. Virus Res. 68 (1), 71–85. Larsen, D.L., Karasin, A., Olsen, C.W., 2001. Immunization of pigs against influenza virus infection by DNA vaccine priming followed by killed-virus vaccine boosting. Vaccine 19 (20–22), 2842–2853. Li, G.X., Tian, Z.J., Yu, H., Jin, Y.Y., Hou, S.H., Zhou, Y.J., et al., 2009. Fusion of C3d with hemagglutinin enhances protective immunity against swine influenza virus. Res. Vet. Sci. Vet. Sci. 86 (3), 406–413. Macklin, M.D., McCabe, D., McGregor, M.W., Neumann, V., Meyer, T., et al., 1998. Immunization of pigs with a particle-mediated DNA vaccine to influenza A virus protects against challenge with homologous virus. J. Virol. 2, 1491–1496. Meseda, C.A., Elkins, K.L., Merchlinsky, M.J., Weir, J.P., 2002. Prime–boost immunization with DNA and modified vaccinia virus Ankara vectors expressing herpes simplex virus-2 glycoprotein D elicits greater specific antibody and cytokine responses than DNA vaccine alone. J. Infect. Dis. 186 (8), 1065–1073. Newman, M.J., 2002. Heterologous prime–boost vaccination strategies for HIV-1: augmenting cellular immune responses. Curr. Opin. Investig. Drugs 3 (3), 374– 378. Olsen, C.W., 2002. The emergence of novel swine influenza viruses in North America. Virus Res. 2, 199–210. Ramshaw, I.A., Ramsay, A.J., 2000. The prime–boost strategy: exciting prospects for improved vaccination. Immunol. Today 21 (4), 163–165. Richard, J., Anderson, Joerg Schneider, 2007. Plasmid DNA and viral vector-based vaccines for the treatment of cancer. Vaccine 25S, B24–B34. Robinson, H.L., Boyle, C.A., Feltquate, D.M., Morin, M.J., Santoro, J.C., Webster, R.G., 1997. DNA immunization for influenza virus: studies using hemagglutinin- and nucleoprotein expressing DNAs. J. Infect. 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Recombinant adenovirus encoding the HA gene from swine H3N2 influenza virus partially protects mice from challenge with heterologous virus: A/HK/1/68 (H3N2). Arch. Virol. 11, 2125–2141. Tian, Z.J., Zhou, G.H., Zheng, B.L., Qiu, H.J., Ni, J.Q., Yang, H.L., et al., 2006. A recombinant pseudorabies virus encoding the HA gene from H3N2 subtype swine influenza virus protects mice from virulent challenge. Vet. Immunol. Immunopathol. 111, 211–218. Torres, C.A., Yang, T.K., Mustafa, F., Robinson, H.L., 2000. DNA immunization: effect of secretion of DNA-expressed hemagglutinins on antibody responses. Vaccine 9–10, 805–814. Wang, S., Parker, C., Taaffe, J., Solórzano, A., García-Sastre, A., Lu, S., 2008. Heterologous HA DNA vaccine prime-inactivated influenza vaccine boost is more effective than using DNA or inactivated vaccine alone in eliciting antibody responses against H1 or H3 serotype influenza viruses. Vaccine 26 (29–30), 3626–3633. 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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Prime–boost immunization with HA/C3d DNA followed by a recombinant pseudorabies virus boost enhanced protective immunity against H3N2 swine influenza virus in mice Guo-Xin Li a,b, Yan-Jun Zhou a, Hai Yu a, Zhi-Jun Tian b, Li-Ping Yan a, Qiang Zhang c, Shou-Ping Hu b, Guang-Zhi Tong a,b,* a
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518 Ziyue Road, Minhang District, Shanghai 200241, China Division of Swine Infectious Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 427 Maduan Street, Harbin 150001, Heilongjiang, China c Technical Center for Animals, Plants and Foods Inspection and Quarantine Shanghai Entry-Exit Inspection and Quarantine Bureau PRC, No. 1208 Minsheng Road, Pudong New Area, Shanghai 200135, China b
a r t i c l e
i n f o
Article history: Accepted 15 September 2009
Keywords: Swine influenza virus DNA vaccines C3d Pseudorabies virus Prime–boost
a b s t r a c t DNA and recombinant virus vaccines against swine influenza virus (SIV) have been pursued with promising results, but induce poor immunogenicity. This study evaluated the effects of a vaccine regimen in mice including priming with three DNA vaccines expressing soluble HA (sHA), complete HA (tmHA), or sHA fused with three copies murine C3d (sHA–mC3d3) and boosting with recombinant pseudorabies virus expressing HA (rPRV–HA). Immune responses were monitored by ELISA, HI assays, and virus neutralization. Protective efficacy was evaluated by virus isolation from lungs, distribution in tissues, and pathology following challenge with H3N2 SIV. Priming with sHA–mC3d3 and boosting with rPRV–HA induced higher levels of HA-specific antibodies and yielded the most effective protection. This finding implied that priming with a DNA vaccine expressing C3d fused with antigen and boosting with a recombinant vector vaccine is an effective way to induce protective humoral immunity and prevent some infectious diseases. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Swine influenza virus (SIV) causes severe respiratory disease characterized by an acute explosive outbreak of symptoms such as coughing, high fever, nasal discharge, anorexia, and weight loss (Olsen, 2002). Under field conditions, coinfection of SIV with other pathogens could result in a significant negative economic impact for the swine industry (Choi et al., 2003). Beyond veterinary implications, influenza virus infection in pigs also poses an important public health concern. Swine are referred to as a ‘‘mixing vessel” because of their susceptibility to both human and avian influenza viruses (Ito et al., 1998; Castrucci et al., 1993). Reassortment of avian and mammalian influenza viruses in swine may produce new viruses, some of which may have the potential to transmit to humans (Webster et al., 1992; Karasin et al., 2000). Therefore, it is important to develop effective strategies to control swine influenza to prevent virus replication in the swine ‘‘mixing vessel”,
* Corresponding author. Address: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518 Ziyue Road, Minhang District, Shanghai 200241, China. Tel.: +86 21 54113138; fax: +86 21 54081818. E-mail address: [email protected] (G.-Z. Tong). 0034-5288/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2009.09.005
thus decreasing the possibility of creating potentially pandemic influenza virus reassortments. Inactivated vaccines are commercially available, but new vaccines that are capable of inducing virus-specific neutralizing antibody plus cell-mediated immunity will provide superior protection against acute influenza diseases (Wesley et al., 2004). In recent years, as an alternative to conventional swine influenza vaccines, DNA vaccines and recombinant virus vaccines have been pursued with some positive and promising results (Macklin et al., 1998; Wesley et al., 2004; Endo et al., 1991; Larsen et al., 2001; Tang et al., 2002). Our recent study showed that three repeated inoculations of DNA vaccines expressing different forms of hemagglutinin (HA) elicited specific immune responses and protected mice from homologous SIV challenge. Although the results were promising, the requirement for three injections makes the DNA vaccines more theoretical than practical for use in swine (Li et al., 2009). In another study, we constructed a recombinant pseudorabies virus expressing HA (rPRV–HA) which protected mice from homologous SIV challenge, although only weak antibody responses were induced (Tian et al., 2006). Recombinant virus vaccines are not suitable for booster immunization when priming with vaccines produced in the same vector
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because the immunity induced by prior immunization may neutralize or inactivate the vector and interfere with antigen presentation. The main disadvantage of DNA vaccines is their poor immunogenicity, especially in large animals. Large quantities of DNA are required to induce only modest immunogenicity (Graham et al., 2006). To circumvent the above problems, we explored a combination vaccine strategy. The immune response was primed with an antigen delivered by one vector, and then boosted using the same antigen delivered by an immunologically distinct vector to augment immune response and protection (Newman, 2002). Several studies have shown that a prime–boost immunization regimen with a DNA plasmid and recombinant virus vaccine, both expressing the same antigen, can induce a strong immune response, including cell-mediated immunity (Schneider et al., 1998; Dégano et al., 1999; Amara et al., 2001). Furthermore, a regimen including a DNA prime and inactivated influenza vaccine boost induced stronger immune responses than did the prime– boost using inactivated vaccine or DNA vaccines alone (Larsen et al., 2001; Wang et al., 2008). However, it is unknown whether a DNA vaccine expressing soluble HA, complete HA, or a fusion of HA with a molecular adjuvant is more effective in heterologous prime–boost immunization regimens. The purpose of the present study was to determine if a vaccine strategy including priming with HA-expressing DNA and boosting with rPRV–HA could enhance immune responses and protection efficiency against homologous SIV challenge. Our previous study showed that a DNA vaccine expressing a fusion of soluble HA with three copies of murine C3d (sHA–mC3d3) induced a stronger immune response than a DNA vaccine expressing soluble HA (sHA) or complete HA (tmHA) (Li et al., 2009). This study determined that priming with sHA–mC3d3 was more effective than priming with either sHA or tmHA when boosting with rPRV–HA. 2. Materials and methods 2.1. Viruses and cell cultures SIV strain A/Swine/Heilongjiang/74/2000 (H3N2) (SwHLJ74) was provided by Dr. Li at Harbin Veterinary Research Institute, Harbin, China. PRV Bartha-K61 strain and recombinant pseudorabies virus expressed HA (rPRV–HA) were propagated and titrated in PK-15 or Vero cells as previously described (Tian et al., 2006). All cells were grown and maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U/ml ampicillin, and 100 lg/ml streptomycin. 2.2. HA–DNA vaccines The HA–DNA vaccines encoding complete HA (tmHA), soluble HA (sHA), or a soluble fused form of HA (sHA–mC3d3) were previously constructed from the H3N2 subtype of SIV (Li et al., 2009). tmHA expresses full-length wild-type HA. sHA was generated by deleting the transmembrane and cytoplasmic domains of HA and replacing the tissue plasminogen activator (tPA) leader sequence with a signal peptide. sHA–mC3d3 was generated by inserting three copies of murine C3d downstream of sHA (Fig. 1). All plasmids were amplified in Escherichia coli strain DH5a and were purified using anion-exchange resin columns (Qiagen). 2.3. Immunization of mice Eight-week-old BALB/c female mice were obtained from the Laboratory Animal Center of Harbin Veterinary Research Institute for immunogenicity studies. Animal maintenance and experimental protocols were approved by the Animal Experiment Ethics
Natural leader Transmembran A
tmHA
HA1
HA2
B
sHA
HA1
HA2
tPA leader C
sHA/mC3d3
HA1
Linker HA2
mC3d
mC3d
mC3d
Fig. 1. Schematic representation of DNA vaccine constructs. (A) Structure of the wild-type, transmembrane form of HA. (B) Structure of extracellular part of HA, linked with a signal sequence of tPA encoding a secreted HA (sHA). (C) Structure of sHA linked with three copies of murine C3d (sHA–mC3d3). Two repeats of four glycines and one serine {(G4S)2} as the linkers were inserted at the junctures of HA and mC3d and between each mC3d repeat.
Committee of the authors’ institute. The mice (18 mice per group) received two immunizations at weeks 0 and 4 with different combination immunizations (Table 1). Animals were injected intramuscularly (i.m.) with 100 lg plasmid DNA or inoculated intranasally (i.n.) with 105 PFU of rPRV–HA as indicated in Table 1. Sera were collected at 0, 2, 4, 6, and 8 weeks after the primary immunization to detect specific antibodies. At week 8 after primary immunization, mice were challenged i.n. with 105 TCID50 of SwHLJ74 (H3N2). 2.4. Serological assay An endpoint ELISA was performed to assess the titers of HA-specific antibody. Purified influenza virus was used to coat plates as described previously (Chen et al., 2007). Endpoint dilution titers from immunized mice that were twofold higher than sera from mice in control groups were considered positive. The hemagglutinin inhibition (HI) assay was conducted as previously described (Robinson et al., 1997) using four hemagglutination units of SIV virus. Neutralization assays were conducted as previously reported (Torres et al., 2000). Neutralization titers were reported as the highest dilution giving complete inhibition of replication of MDCK cells given a TCID50 of 100 in 50 ll DMEM medium. The presence of replicating virus in a well was scored by hemagglutination. 2.5. Lymphocyte proliferative responses Lymphocyte proliferative responses using mouse splenocytes were detected as previously described (Li et al., 2009). Briefly, four weeks after boost immunization splenocytes were isolated from immunized mice, suspended in RPMI 1640, and seeded into 96well plates at 4 105 cells per well (200 ll). The cultures were stimulated with either Con A (positive control), 20 ll of inactivated and purified swine influenza virus as the specific antigen, or nothing (negative control). The proliferative response was measured by adding 20 ll WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) to each well with a further 5 h of incubation. The stimulation index was calculated as the ratio of the average OD values in wells containing antigen-stimulated cells to the average OD of wells containing only cells with medium. 2.6. IL-4 and IFN-c release assay At week 4 after boost immunization, the spleens from three mice from each group were harvested. Mouse splenocytes were prepared as described (Bounous et al., 1992) and incubated at 37 °C in 96-well plates at a concentration of 2 105 cells per well in the presence of 20 ll inactivated and purified SIV. After 72 h incubation, supernatants were harvested and the presence of IL-4
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G.-X. Li et al. / Research in Veterinary Science 88 (2010) 345–351 Table 1 Vaccination schedule and challenge of mice. Immunization and challenge
Groups sHA
tmHA
sHA/mC3d3
sHA + rPRV–HA
tmHA + rPRV–HA
sHA/mC3d3 + rPRV–HA
rPRV–HA
Control
Prime (week 0) Boost (week 4)
sHA sHA
tmHA tmHA
sHA/mC3d3 sHA/mC3d3
sHA rPRV–HA
tmHA rPRV–HA
sHA/mC3d3 rPRV–HA
Vector rPRV–HA
Vector Vector
Challenge (week 8)
Challenged with SwHLJ74 (H3N2)
and IFN-c was tested with mouse IL-4 and IFN-c ELISA kits (Biosource, USA) according to the manufacturer’s instructions. The concentrations of mouse IL-4 and IFN-c in the samples were determined from the standard curves.
3. Results
2.7. Virus detection and isolation
Levels of anti-HA IgG responses in immunized mice were measured by ELISA. At week 2 after the first inoculation, the antibody titer exceeded 1:200 in all groups immunized with HA–DNA, while the antibody titer of control group mice was undetectable. At week 4 after boost immunization, the anti-HA IgG titer elicited by sHA– mC3d3 in the DNA prime–boost groups was significantly higher than the titers elicited by sHA and tmHA (P < 0.01, Fig. 2). Furthermore, the DNA prime–rPRV–HA boost elicited higher anti-HA IgG titers compared with the corresponding DNA prime–boost groups or the rPRV–HA only group (Fig. 2). The antibody titers elicited by sHA prime–rPRV–HA boost and tmHA prime–rPRV–HA boost were similar. Interestingly, sHA–mC3d3 prime–rPRV–HA boost elicited the highest anti-HA IgG titers among these groups. The differences in the anti-HA IgG levels between sHA–mC3d3 prime– rPRV–HA boost and any other DNA prime–rPRV–HA boost was statistically significant (P < 0.01, Fig. 2).
Five mice in each group were euthanized 3, 7, and 10 days postchallenge (DPC). At necropsy, lung, spleen, kidney, and cerebrum samples were collected and analyzed by immunofluorescence assay (IFA) (Yoon et al., 1992). Nine- to eleven-day-old embryonated chicken eggs were inoculated allantoically with 0.2 ml of the lung samples and incubated at 37 °C for 72 h. The allantoic fluids were then harvested and tested for hemagglutinin activity. Positive samples were further characterized by the HI test. 2.8. Histopathology Thin sections of formalin-fixed, paraffin-embedded tissues were taken from the lungs, spleen, kidney, and cerebrum and stained using hematoxylin–phloxin–safran (HPS) as described previously (Dea et al., 1991). 2.9. Statistical analysis All data were presented as the mean ± standard error (SE). The presence of statistical differences among groups was determined by one-way ANOVA; the method of least significant difference (LSD or Dunnett T 3 test) was used to compare the effects among groups with the aid of SPSS 13.0 for windows. Differences were deemed statistically significant when the P-value was <0.05.
sHA
3.2. Hemagglutinin–inhibition (HI) and virus neutralization (VN) titers The levels of functional antibodies in sera from immunized mice were measured using hemagglutination inhibition and microneutralization assays (Figs. 3 and 4). At week 4 after boost immunization, HI and VN titers elicited by sHA–mC3d3 in the DNA prime–
tmHA+rPRV-HA
tmHA
sHA/mC3d3+rPRV-HA
sHA/mC3d3
rPRV-HA
sHA+rPRV-HA
Control
sHA
tmHA+rPRV-HA
tmHA
sHA/mC3d3+rPRV-HA
sHA/mC3d3
rPRV-HA
sHA+rPRV-HA
Control
250 d 200
HI antibody titers
Endpoint dilution titers
100000
3.1. Antibody responses to prime–boost immunizations detected by ELISA
10000
1000
c 150
bc
bc
b 100 b
b
50 a
< 100 0
2 4 6 Weeks after primary immunization
8
Fig. 2. Anti-HA IgG levels in mice after vaccination. Mice were primed at week 0 and boosted at week 4. Sera were collected from five mice per group for determination of specific IgG levels by ELISA.
0 Fig. 3. The hemagglutination inhibition (HI) antibody responses in sera immunized with DNA vaccines, rPRV–HA, or the combination of both. The HI antibody titers are shown as the geometric means for each group (five mice) with standard errors. Means not sharing a common letter are significantly different at P < 0.05.
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sHA
tmHA+rPRV-HA
tmHA
sHA/mC3d3+rPRV-HA
sHA/mC3d3
rPRV-HA
sHA+rPRV-HA
Control
sHA tmHA sHA/mC3d3 sHA+rPRV-HA
tmHA+rPRV-HA sHA/mC3d3+rPRV-HA rPRV-HA Control
2.6
6000 2.4
c 5000
NAb titers
4000
Stimulation index
2.2
b
3000 2000 1000
2 1.8 1.6 1.4
a a
a
a 1.2
a a
0 Fig. 4. The neutralizing antibody (NAb) responses in sera immunized with DNA vaccines, rPRV–HA, or the combination of both. The NAb titers against H3N2 virus infection of MDCK cells are shown as the geometric means from each group (five mice) with standard errors. Means not sharing a common letter are significantly different at P < 0.05.
boost groups were significantly higher than the titers raised by sHA and tmHA (P < 0.05; Figs. 3 and 4). The results indicated that C3d was able to stimulate specific B cells to increase the production of antibodies to SIV. Furthermore, the DNA prime–rPRV–HA boost elicited higher levels of HI and VN when compared to the corresponding DNA prime–boost regimens or rPRV–HA alone. Particularly, the sHA–mC3d3 prime–rPRV–HA boost elicited the highest HI and VN titers among these groups.
1 Fig. 5. Lymphocyte proliferative responses at week 4 after boost immunization. The splenocytes were isolated and after 72 h of stimulation with SIV, WST-8 was added. The OD value determined after further 5 h incubation. Data represent the means plus standard errors.
duced by sHA or tmHA. In contrast, tmHA induced a numerically higher level of IFN-c compared with sHA and sHA–mC3d3. The results indicated that C3d fusion elicited a Th2-biased immune response by inducing IL-4 production while the full-length form of HA (tmHA) tended to produce a Th1-biased immune response. Furthermore, the DNA prime–rPRV–HA boost elicited a higher level of IL-4 and IFN-c compared with the corresponding DNA prime– boost groups or the rPRV–HA group (Fig. 6). Interestingly, the sHA–mC3d3 prime–rPRV–HA boost induced the highest level of IL-4 while the tmHA prime–rPRV–HA boost induced the highest level of IFN-c (Fig. 6).
3.3. Lymphocyte proliferation assay
3.5. Virus distribution after challenge
Lymphocyte proliferative responses for three mice from each group were analyzed at week 4 after boost immunization. Proliferation responses in all seven groups immunized with HA–DNA, rPRV–HA, or the combination of both were higher than those of the controls (Fig. 5). For the DNA prime–boost groups, the stimulation index of mice injected with tmHA was higher than that of mice injected with sHA or sHA–mC3d3 (Fig. 5). Furthermore, the DNA prime–rPRV–HA boost elicited increased cell proliferation compared with the corresponding DNA prime–boost groups or the rPRV–HA only group. The proliferation responses elicited by the sHA prime–rPRV–HA boost and the sHA–mC3d3 prime–rPRV–HA boost were similar. However, the tmHA prime–rPRV–HA boost elicited the highest level of proliferative responses among these groups (Fig. 5).
The distribution of SIV antigens in different tissues of immunized animals at necropsy was examined by IFA. Many SIV antigens were detected in the lungs of control animals from 3 to 10 DPC. Less virus antigen was detected in the lungs of DNA or rPRV–HA immunized mice 3 DPC and no virus antigens were detected 7 or 10 DPC. Moreover, fewer virus antigens were detected in the lungs of mice with DNA prime–rPRV–HA boost compared with the mice with DNA prime–boost or the rPRV–HA only 3 DPC (Table 2). SIV antigens could be detected in the spleens of control mice 3 DPC, but not in those of the DNA or rPRV–HA inoculated mice (Table 2).
3.4. Cytokine production by splenocytes from vaccinated mice At week 4 after boost immunization, the spleens from three mice from each group were harvested. Splenocytes from each group were isolated, restimulated with inactivated swine influenza virus, and assessed for the presence of IFN-c, an indication of a Th1 response, and IL-4, an indication of a Th2 response. Mice immunized with HA–DNA, rPRV–HA, or the combination of both showed a rise in IL-4 and IFN-c (Fig. 6). For the DNA prime-boost groups, the level of IL-4 induced by sHA–mC3d3 was higher than that in-
3.6. Isolation of challenge virus from lungs In general, virus was isolated from fewer mice in the DNA prime–rPRV–HA boost groups than that of mice in the corresponding DNA prime–boost groups. At 3 DPC, virus was isolated from all control mice, but no virus was isolated from mice from the sHA– mC3d3 prime–rPRV–HA boost group. The percentage of mice from which virus was isolated dropped in the sHA group (2/5) and tmHA group 2 (1/5) at day 7 post-challenge, and no virus was isolated from mice from the other immunized groups. By 10 DPC, virus could be isolated from only 3/5 control mice, and no virus was isolated from the DNA or rPRV–HA immunized groups (Table 3). These results indicated that immunization with DNA, rPRV–HA, or a combination of the two strategies was able to inhibit replica-
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sHA tmHA sHA/mC3d3 sHA+rPRV-HA
A
tmHA+rPRV-HA sHA/mC3d3+rPRV-HA rPRV-HA Control
Table 3 Isolation of swine influenza virus from the lungs of immunized mice following challenge with SwHLJ74.
1400 1200 1000
pg/ml
800
a
Groups
3 DPC
7 DPC
10 DPC
sHA tmHA sHA/mC3d3 sHA + rPRV–HA tmHA + rPRV–HA sHA/mC3d3 + rPRV–HA rPRV–HA Control
4/5a 4/5 1/5 2/5 1/5 0/5 2/5 5/5
2/5 1/5 0/5 0/5 0/5 0/5 0/5 5/5
0/5 0/5 0/5 0/5 0/5 0/5 0/5 3/5
Number of mice with positive results/total mice.
600 tion of homologous SIV to a great extent and sHA–mC3d3 prime– rPRV–HA boost was the most effective at inhibiting virus replication among these immunization regimens.
400 200
3.7. Pathological findings after challenge
0
B
IL-4 sHA tmHA sHA/mC3d3 sHA+rPRV-HA
tmHA+rPRV-HA sHA/mC3d3+rPRV-HA rPRV-HA Control
1600 1400 1200
pg/ml
1000 800 600 400
Microscopic lesions were observed in most of the control group mice following SIV challenge. Observed lesions included thickened alveolar septa and infiltration of lymphocytes leading to mild to severe interstitial pneumonia (Fig. 7), collapse of follicles, depletion of germinal centers, blurred white medulla, hyperplastic red medulla in spleens (Fig. 7), glial cell nodule formation and liquefaction in the cerebra, slight stroma hyperplasy, and lymphocyte infiltration in kidneys (Table 4). In most of the immunized groups, a few mice showed mild interstitial pneumonia, characterized by slightly thickened alveolar septa, while no pathological lesions were found in the kidneys or cerebra (Table 4). Furthermore, most immunized mice had follicle hyperplasia and germinal center enlargement in spleens, possibly indicative of an immune response to the vaccination. Interestingly, in the sHA–mC3d3 prime–rPRV– HA boost group, none of mice showed microscopic lesions in the lungs, spleens, kidneys, or cerebrum (Table 4). These results indicated that DNA prime–rPRV–HA boost, especially the sHA– mC3d3 prime–rPRV–HA boost, yielded more effective protection than did DNA prime–boost.
200 0
4. Discussion
IFN-gamma
Fig. 6. Levels of IL-4 (A) and IFN-c (B) from splenocytes in mice immunized with DNA vaccines, rPRV–HA, or the combination of both. At week 8 after primary immunization, splenocytes from each mouse group were harvested and were cultured at 2 105 cells per well in 96-well plates and stimulated with inactivated swine influenza virus. IFN-c and IL-4 production in the supernatant was analyzed by ELISA. Data represent the means plus standard errors.
DNA immunization is a promising vaccine strategy against infectious diseases. However, poor immunogenicity has been an obstacle for the clinical application of DNA vaccines. In order to circumvent this problem, various strategies, such as adjuvant augmentation and delivery system optimization, have been considered (Richard and Anderson, 2007). In our previous study, the C3d molecular adjuvant fused with SIV hemagglutinin (sHA–
Table 2 The distribution of swine influenza virus in different organs of immunized mice on different days following challenge with SwHLJ74. Groups
sHA tmHA sHA/mC3d3 sHA + rPRV–HA tmHA + rPRV–HA sHA/mC3d3 + rPRV–HA rPRV–HA Control
3 DPC
7 DPC
10 DPC
Lungs
Spleens
Lungs
Spleens
Lungs
Spleens
+++ +++ +++ ++ ++ ++ +++ ++++
– – – – – – – +
– – – – – – – ++++
– – – – – – – –
– – – – – – – ++
– – – – – – – –
Tissues were collected from all mice tested, with two frozen sections for each tissue, and examined by indirect immunofluorescence antibody test: (–) no stained cells in a whole section; (+) 1–2 positive-stained cells in a section; (++) 1–2 stained cells in one view field; (+++) 2–10 stained cells in one view field; (++++) >10 stained cells in one view field.
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Fig. 7. Histological lesions of mice in sHA–mC3d3 prime–rPRV–HA boost (A–B) and control groups (C–D) 7 days post-challenge with SwHLJ74. (A) Spongiform aspect of the lungs, showing well-delineated, thin alveolar septa with the normal aspect of a small blood vessel. (B) Lymph follicle hyperplasia in spleen. (C) Interstitial pneumonia with alveolar septa thickened by lymphocyte infiltration. (D) Collapsed follicle in spleen. H&E staining. Magnification 40.
Table 4 Gross lesions of mice at necropsy after challenge with SwHLJ74. Groups
Lungs
Kidneys
Cerebrum
Spleen
sHA tmHA sHA/mC3d3 sHA + rPRV–HA tmHA + rPRV–HA sHA/mC3d3 + rPRV–HA rPRV–HA Control
7/15a 6/15 4/15 4/15 3/15 0/15 4/15 10/15
0/15 0/15 0/15 0/15 0/15 0/15 0/15 7/15
0/15 0/15 0/15 0/15 0/15 0/15 0/15 5/15
0/15 0/15 0/15 0/15 0/15 0/15 0/15 9/15
Tissue lesions were scored as: lung, interstitial pneumonia with alveolar septa thickened by infiltration of lymphocytes and macrophages surrounding bronchiole; spleen, collapsed follicles; cerebrum, glial cell nodule formation or liquefaction in cerebrum; kidney, slight stroma hyperplasia and infiltration of lymphocytes. a Number of mice showing pathological lesions/total mice.
mC3d3) enhanced protective immunity against SIV in mice (Li et al., 2009). Large doses of DNA (three inoculations, total 300 lg) were given to induce strong immune responses in that experiment. However, multiple doses may make this vaccine strategy difficult to implement in swine in practice. Thus, in the current study, we investigated the effects of fewer doses (two inoculations, total 200 lg) of DNA inoculation. The results showed that two inoculations of sHA–mC3d3 induced HA-specific immune responses and protected mice from homologous SIV challenge. As such, in future studies, it is important to assess the effects of the DNA vaccine in the context of further decreasing doses or times of injection. In the current study, we compared the levels of immune response and protection efficiency between homologous (DNA + DNA or rPRV–HA alone) and heterologous (DNA + rPRV–HA) prime–boost vaccination strategies. DNA has been considered an
effective priming vector because of the long lasting expression period and the induction of cellular and/or humoral immunity (Ramshaw and Ramsay, 2000). Several recombinant vectors have been developed with the aim of inducing the prolonged expression of high levels of viral antigens, such as adenovirus, pseudorabies virus, fowlpox virus, semliki forest virus, and vesicular stomatitis virus (Richard and Anderson, 2007). However, immune responses elicited by these recombinant virus vaccines targeted not only the foreign antigens but also the vector itself, and pre-existing immunity to the vector has been shown to dramatically decrease vaccination efficiency (Sharpe et al., 2001). DNA prime and recombinant viral vector boost immunization regimens appear to be very effective at inducing high levels of immune responses (Schneider et al., 1998; Dégano et al., 1999; Meseda et al., 2002; Dory et al., 2006). In this study, heterologous DNA prime and rPRV–HA boost in mice elicited higher levels of protective antibodies and yielded more effective protection compared to homologous DNA prime– boost immunization regimens or the single rPRV–HA immunization. However, studies with heterologous prime–boost vaccine regimens in the past have provided conflicting results. Breathnach’s research into influenza vaccines showed that recombinant modified vaccinia virus Ankara (rMVA) induced protective immunity in horses independent of a DNA prime (Breathnach et al., 2006), while a heterologous prime–boost immunization regimen with DNA and rMVA was effective in mice (Dégano et al., 1999). The differences observed in these studies may be partly attributed to differences in the animal models. Therefore, it is important to evaluate the effects of DNA prime–rPRV–HA boost immunization regimens and single rPRV–HA immunization in swine in future studies. Although a previous study indicated that the DNA prime may be more effective than the inactivated influenza vaccine in priming
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the immune system (Wang et al., 2008), in this study sHA–mC3d3 prime–rPRV–HA boost induced higher levels of HA-specific antibodies compared to either sHA or tmHA prime and rPRV–HA boost. However, it is possible that DNA immunization is more effective in eliciting better and potentially longer lasting HA-specific B cell memory (Wang et al., 2008). Low-dose antigen delivery has been shown to be more effective in eliciting better antibody responses and B cell memory (González-Fernández and Milstein, 1998; Bot et al., 1997). Therefore, DNA vaccines may be more suitable as priming vectors than boosting vectors. Interestingly, our results suggested that fusion of C3d with HA may be more effective as a priming immunogen compared to the non-fusion form HA. This result was supported in a previous study that found fusion of C3d with HA may induce affinity maturation of the antibody response and elicit memory B cells more effectively than non-C3d fused forms of HA (Ross et al., 2000). Therefore, the fusion of C3d with antigen priming followed by boosting with other vaccines, such as recombinant vector vaccines or inactivated vaccines, may be an attractive solution to inducing humoral protective humoral immunity to some infectious diseases. In future studies there are a number of areas which should be explored, including the mucosal immune response induced by these vaccination regimens and evaluation of the effects of priming with various doses of DNA. Furthermore, since H1N1 and H3N2 are the dominant subtypes causing swine influenza worldwide and can be found co-circulating in the same herd with some frequency, it is important to determine whether such prime–boost regimens are able to provide protective immunity against heterologous strains of SIV. Acknowledgements The study was supported by Grants from the National Basic Research Program (973) of China (No. 2005CB523200), the National High-Tech Research and Development Program (863 Program) of China (No. 2006AA10A204) and the National Scientific Supporting Program of China (No. 2006BAD06A04/03/01). References Amara, R.R., Villinger, F., Altman, J.D., Lydy, S.L., O’Neil, S.P., Staprans, S.I., et al., 2001. Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science 292 (5514), 69–74. Bot, A., Antohi, S., Bona, C., 1997. Immune response of neonates elicited by somatic transgene vaccination with naked DNA. Front. Biosci. 2, d173–d188. Bounous, D.I., Campagnoli, R.P., Brown, J., 1992. Comparison of MTT colorimetric assay and tritiated thymidine uptake for lymphocyte proliferation assays using chicken splenocytes. Avian. Dis. 4, 1022–1027. Breathnach, C.C., Clark, H.J., Clark, R.C., Olsen, C.W., Townsend, H.G., Lunn, D.P., 2006. Immunization with recombinant modified vaccinia Ankara (rMVA) constructs encoding the HA or NP gene protects ponies from equine influenza virus challenge. Vaccine 24 (8), 1180–1190. Castrucci, M.R., Donatelli, I., Sidoli, L., Barigazzi, G., Kawaoka, Y., Webster, R.G., 1993. Genetic reassortment between avian and human influenza A viruses in Italian pigs. Virology 193 (1), 503–506. Chen, Y., Xin, X.G., Yang, H.L., Li, T., Li, H.Y., Qiao, C.L., et al., 2007. Establishment of an indirect ELISA for detection antibodies against swine influenza virus. Prog. Vet. Med. 4, 308–311 (in Chinese). Choi, Y.K., Goyal, S.M., Joo, H.S., 2003. Retrospective analysis of etiologic agents associated with respiratory diseases in pigs. Can. Vet. J. 44 (9), 735–737. Dea, S., Bilodeau, R., Sauvageau, R., Martineau, G.P., 1991. Outbreaks in Quebec pig farms of respiratory and reproductive problems associated with encephalomyocarditis virus. J. Vet. Diagn. Invest. 3 (4), 275–282. Dégano, P., Schneider, J., Hannan, C.M., Gilbert, S.C., Hill, A.V., 1999. Gene gun intradermal DNA immunization followed by boosting with modified vaccinia virus Ankara: enhanced CD8+ T cell immunogenicity and protective efficacy in the influenza and malaria models. Vaccine 18 (7–8), 623–632. Dory, D., Fischer, T., Béven, V., Cariolet, R., Rziha, H.J., Jestin, A., 2006. Prime–boost immunization using DNA vaccine and recombinant Orf virus protects pigs against Pseudorabies virus (Herpes suid 1). Vaccine 24 (37–39), 6256–6263.
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