Immune responses and protective efficacy of a recombinant swinepox virus expressing HA1 against swine H1N1 influenza virus in mice and pigs

Vaccine 30 (2012) 3119–3125

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Immune responses and protective efficacy of a recombinant swinepox virus expressing HA1 against swine H1N1 influenza virus in mice and pigs Jiarong Xu a , Dongyan Huang a,b , shichao Liu a , Huixing Lin a , Haodan Zhu a , Bao Liu a , Chengping Lu a,∗ a b

College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, China

a r t i c l e

i n f o

Article history: Received 28 November 2011 Received in revised form 27 January 2012 Accepted 10 February 2012 Available online 3 March 2012 Keywords: Swine H1N1 influenza virus Hemagglutinin Recombinant swinepox virus Animal trial Immunogenicity Protective efficacy

a b s t r a c t Swine influenza virus (SIV) is not only an important respiratory pathogen in pigs but also a potent threat to human health. Although immunization with recombinant poxviruses expressing protective antigens as vaccines has been widely used for against many infectious diseases, development of recombinant swinepox virus (rSPV) vector for the purpose has been less successful. Here, we report the construction of a recombinant swinepox virus (rSPV-HA1) expressing hemagglutinin (HA1) of H1N1 SIV. Immune responses and protection efficacy of the vaccination vector were evaluated in both the mouse model and the natural host: pig. Prime and boost inoculations of rSPV-HA1 yielded high levels of neutralization antibody against SIV and elicited potent H1N1 SIV-specific IFN-␥ response from T-lymphocytes. Complete protection of pigs against H1N1 SIV challenge was observed. No pigs showed evident systemic and local reactions to the vaccine and no SIV shedding was detected from pigs vaccinated with rSPV-HA1 after challenge. Our data demonstrated that the recombinant swinepox virus encoding HA1 of SIV H1N1 may serve as a promising SIV vaccine for protection against SIV infection. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Swine influenza (SI) is an acute and highly contagious respiratory disease caused by type A influenza viruses. SI is characterized by sudden onset, coughing, respiratory distress, weight loss, fever, nasal discharge and rapid recovery and featured with high morbidity and low mortality [1,2]. Due to the segmented genome, influenza A viruses undergo infrequent antigenic shift or reassortment, leading to generation of new variant viruses. Porcine lung epithelial cells possess receptors for both avian and mammalian influenza viruses [3,4]. Therefore, pigs have been considered as the reservoir host or “mixing vessel” for the generation of new recombinant strains with pandemic capacity [5]. The gene of RNA segment 4 of swine influenza virus (SIV) encodes the large hemagglutinin (HA) glycoprotein which is considered to be the main immune antigen [6–9]. HA can be cleaved into HA1 and HA2 glycoproteins with HA1 as the most antigenic in induction of host immune response [10–13]. A number of studies have demonstrated that immunization with recombinant HA is capable of inducing both cell-mediated and humoral immunity [6]. Swinepox virus (SPV) is known to infect porcine species only [14], causes mild clinical symptoms with localized skin lesions

∗ Corresponding author. Tel.: +86 2584396517; fax: +86 2584396517. E-mail address: [email protected] (C. Lu). 0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2012.02.028

that heal naturally [15]. Poxviruses have potential to induce both humoral and cellular immune response [16,17]. In addition, laboratory manipulation of poxvirus DNA for various applications has become almost standard practice, making it easy to develop recombinant vaccines based on poxviruses [18,19]. Currently, the only available vaccines for SI are inactivated whole virus by intramuscular administration. Application of these vaccines reduces the severity of disease but does not provide consistent protection from infection. Although several recombinant virus vaccines have been developed against SIV [2,20–22], no commercial live SI vaccine is available against SIV as the fowlpox virus (FPV) vector vaccine is available against avian influenza virus [6,23]. In this study we have developed a recombinant swinepox virus (rSPV) encoding HA1 of SIV H1N1 and evaluated its safety, immunogenicity and protective efficacy in mice and pigs.

2. Materials and methods 2.1. Viruses and cells Wild type swinepox virus (wtSPV, Kasza strain, ATCC VR363) and porcine kidney cells (PK-15, ATCC CCL-33) were purchased from the American Type Culture Collection. A crude viral stock was prepared and the recombinant SPV and wtSPV titers were determined as described previously [17]. The H1N1 SIV (A/swine/Shanghai/1/2005), gift of Dr. Xian Qi, was propagated only

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2.6. Immunofluorescence assays

A LF

lacZ

RF

AP r Indirect immunofluorescence assay (IFA) was performed as described previously [17,24]. The rSPV-HA1, wtSPV-infected and non-infected PK-15 cells were incubated with H1N1 SI convalescent positive serum (1:1000 dilution). Then, the cells were stained with Staphylococcal protein A-FITC (Boshide, Wuhan, China). After a final wash, all wells were examined by fluorescence microscopy (Zeisss, Germany).

BamHI

pUSZ11 8391 bp

B

p11

H A1

LF

lacZ

RF

APr 2.7. Animal experiment

BamHI BamHI

pUSZ11/HA1

9422 bp Fig. 1. Schematic construction of H1N1 SIV HA1 recombinant SPV transfer vector pUSZ11/HA1. (A) Swinepox virus vector pUSZ11. (B) Recombinant SPV HA1 transfer vector pUSZ11/HA1. LF: left flanking region; RF: right flanking region; APr : antipenicillin gene.

once in specific pathogen-free embryonated eggs (Nanjing veterinary drug and instrument factory, Nanjing, China). Madin-Darby canine kidney cells (MDCK) were used for SIV isolation and titration assay. All experiments involving H1N1 SIV were conducted using biosafety level 3 procedures. 2.2. Construction of recombinant swinepox virus plasmid HA1 gene (containing the promoter P11 sequence) of H1N1 SIV (GenBank accession number EU502884) was chemically synthesized by Invitrogen Biotechnology Co. Ltd. (Shanghai, China), and cloned in pUC19 first and then cloned into BamHI site of pUSZ11 (Fig. 1A) [17]. The correct insertion of HA1 gene in pUSZ11 was confirmed by DNA sequencing with specific primers (F: 5 -gttataggtacccggggatccagtagaatttc-3 ; R: 5 -ctatttgggggatccttattatctagattg-3 ), which we designated as pUSZ11/HA1 (Fig. 1B). 2.3. Generation and screening of the recombinant swinepox virus A pre-confluent monolayer of PK-15 cells grown in a 60 mm diameter plate was infected with wtSPV (0.02 moi) for 2 h, and subsequently transfected with 8 ␮g of the pUSZ11/HA1 plasmid using lipofectamine 2000 (Invitrogen, Shanghai, China). Generation and screening of recombinant swinepox viruses were carried out as described previously [17]. Blue foci isolation was repeated for 5–6 rounds until all foci in a given well were stained blue. The growth kinetics of the recombinant SPV was determined and compared with wtSPV. The recombinant SPV bearing HA1 of SIV H1N1 was designated as rSPV-HA1. 2.4. PCR analysis of the recombinant swinepox virus The SDS-Protease K-Phenol method was used to extract the rSPV-HA1 genomic DNA from the PK-15 cells infected with rSPVHA1. The wtSPV genomic DNA from PK-15 cells infected with wtSPV was used as a negative control. Amplifications were performed with DNA polymerase (Promega, Shanghai, China) using primers described above. 2.5. Western blot The western blot was carried out as described previously [17], with a H1N1 SI convalescent positive serum which had a known neutralizing antibody titer (1:1000 dilution).

All experimental protocols involving mice and pigs were approved by the Laboratory Animal Monitoring Committee of Jiangsu Province. 2.7.1. Mouse model Fifty-four 7-week-old female BALB/c mice were purchased from the Animal Center of Nanjing Army Hospital (Nanjing, China) and randomly divided into three groups (18 mice per group). Groups 1 and 2 were inoculated with rSPV-HA1 or wtSPV, respectively, at the same dose of 0.2 × 107.0 TCID50 in 0.2 ml EMEM and applied over the four legs. Group 3 was inoculated with 0.2 ml EMEM. All inoculations were administered intramuscularly three times at 1, 21 and 35 days post inoculation (dpi). At 21, 35 and 42 dpi, five mice of each group were euthanized and serum samples were obtained for detection of the antibody against H1N1 SIV. At the same time, the lymphocytes were isolated from the spleens for measurement of the H1N1 SIV-specific T lymphocyte proliferation responses. In addition, the supernatants of the lymphocytes stimulated with purified H1N1 SIV HA1 antigen at 35 dpi were obtained for evaluation of the Th1-type cytokine IFN-␥ and Th2-type cytokine IL-4. 2.7.2. Swine experiment Eighteen Yorkshire and Landrace crossbred pigs (seronegative for SIV, SPV and porcine reproductive and respiratory syndrome virus) were obtained from Kangle farm (Changzhou, China). The pigs were weaned at the age of 3 weeks, then delivered to the Animal Disease Research Center of Agriculture Institute of Jiangsu Province and allowed to acclimate to their new environment and new feed for 2 weeks. Fifteen of them were randomly assigned to three groups (5 pigs per group). Three pigs were included as environmental controls (unvaccinated and unchallenged). Each group was housed separately in an individual specified pathogen-free isolation room. At 5 weeks of age, groups 1 and 2 were individually inoculated intramuscularly in the neck area with rSPV-HA1 or wtSPV at 1 × 107.0 TCID50 in 1 ml EMEM per pig. Group 3 was inoculated with 1 ml EMEM. Pigs were closely monitored daily within the first 7 day post-vaccination. At 21 dpi, the serum was collected from each pig to detect neutralizing antibodies (NA) against H1N1 SIV and the peripheral blood mononuclear cells (PBMC) were isolated to evaluate H1N1 SIV-stimulated production of IFN-␥ and IL-4. Then all pigs were challenged with 2 × 105.0 TCID50 H1N1 SIV per pig by nasal inoculation. Clinical signs including hyponoia, inappetence, puffiness of the eyes, snorting, labored breathing, coughing, erythrosis and dry stool were recorded by observing the pigs for 5 days twice per day. Each clinical symptom was given a numerical score of 0 for no sign and 1 for appreciable sign. Finally, clinical sign scores were calculated as the sum of all clinical symptom scores of each pig divided by 2 × 5. Meanwhile, daily rectal temperatures were examined for 5 days post challenge (dpc). Nasal swabs from each pig were collected daily from 0 through 5 dpc to assess virus shedding by titration on MDCK cells. At 5 dpc all of the pigs were humanely euthanized. Lungs were examined for gross and microscopic lesions. The degree

J. Xu et al. / Vaccine 30 (2012) 3119–3125

of consolidation on the surfaces of the seven lung lobes of each lung was estimated visually. Lung pathological scores were calculated as the sum of percent consolidation of each lung lobe divided by 7. Four lung tissue samples from each pig were collected to examine virus presence.

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2.13. Statistical analysis Data were analyzed using two-way ANOVA (a single-factor analysis of variance) and values of P < 0.05 were considered significant. 3. Results

2.8. Indirect ELISA (iELISA) 3.1. Characterization of the recombinant swinepox virus The purified recombinant H1N1 SIV HA1 protein, expressed in E. coli, was used to coat 96-well plates at the concentration of 1 ␮g/ml. The iELISA was carried out as described previously. The sera from the mice inoculated with EMEM were used as the negative control. The results are expressed as OD450nm values. 2.9. Assay for neutralizing antibodies Assay for neutralizing antibodies was carried out as described previously [2]. The titers of neutralizing antibodies were expressed as the highest serum dilution in which no CPE was observed. 2.10. T lymphocyte proliferation assay T lymphocyte proliferation assay was carried out as described previously [25]. Cultures were either stimulated with purified H1N1 SIV HA1 antigen at a final concentration of 10 ␮g/ml or not stimulated. T lymphocyte proliferation is reported as stimulation index. 2.11. Cytokine assay Cytokine assay was carried out as described previously [25] and purified H1N1 SIV HA1 antigen was used as stimulated antigen at a final concentration of 10 ␮g/ml.

As shown in Fig. 2A, an approximately 1.0 kb HA1 gene fragment, which encodes most antigen epitopes of HA glycoprotein, was amplified from the recombinant virus, but not wtSPV. Moreover, the recombinant swinepox virus was confirmed by blue foci in plaque assays. After plaque purification for five times, the TCID50 of rSPV-HA1 was determined to be 101.1 , 103.2 and 107.0 TCID50 /ml, and the TCID50 of wtSPV to be 101.2 , 103.2 and 107.1 TCID50 /ml on PK-15 cells, at 2, 4 and 6 days after infection, respectively. Western blot analysis showed a specific protein band of ∼40 kDa in the cell lysates infected with rSPV-HA1 (Fig. 2B). The 40 kDa molecular weight is consistent with the predicted size of the HA1 protein of H1N1 SIV. The expression of HA1 was also demonstrated in infected cells by IFA (Fig. 2C). Thus, we were confident that the recombinant SPV-HA1 virus efficiently expresses HA1 protein. 3.2. rSPV-HA1 induces humoral immune response in mice rSPV-HA1 induced a moderate level of H1N1 SIV-specific IgG at 21 dpi as shown in Fig. 3A. The second vaccination led to a slightly increase and the third vaccination resulted in a considerable increase in the level of H1N1 SIV-specific IgG. Persistent high levels of neutralizing antibodies (Fig. 3B) were detected in the rSPV-HA1 group with a mean titer of 1:38.4 at 42 dpi. 3.3. rSPV-HA1 induces cell-mediated immune responses in mice

2.12. SIV isolation from lungs and nasal swabs Presence of virus in the lungs and nasal swabs was determined by the appearance of CPE of H1N1 SIV on MDCK cell cultures as described previously [21]. Four lung samples from each pig were collected for the assay: right and left apical and cardiac lung lobes [23]. H1N1 SIV titrations of nasal swabs were carried out as described previously [21]. The titer was calculated using the Reed–Muench statistical method.

As shown in Fig. 4A, high level of proliferation stimulation index (SI 3.86) was observed in the group inoculated with rSPV-HA1 at 42 dpi, which is significantly higher (P < 0.001, n = 5) than the control mice at 21, 35 and 42 dpi. At 35 dpi, the levels of IL-4 produced by PBMC from the EMEM, wtSPV and rSPV-HA1 inoculated groups were 39.97 pg/ml, 40.63 pg/ml and 117.4 pg/ml, respectively. Similarly, the IFN-␥ concentrations were 45.62 pg/ml, 55.74 pg/ml and 175.7 pg/ml

Fig. 2. Characterization of the recombinant swinepox virus. (A) PCR analysis of the recombinant virus rSPV-HA1. Lane 1: rSPV-HA1; Lane 2: wtSPV; Lane 3: DL2000 DNA marker. (B) Expression identification of rSPV-HA1 by Western blot analysis. Lane M: prestained protein marker. Lane 1: rSPV-HA1. Lane 2: wtSPV. Lane 3: non-infected PK-15 cells. The primary antibody was swine convalescent-phase sera and the secondary antibody was Staphylococcal protein A-HRP. (C) Identification of the expression of rSPV-HA1 by IFA on infected PK-15 cells. The primary antibody was swine convalescent-phase sera and the secondary antibody was Staphylococcal protein A-FITC.

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EMEM

2.0

A

B

rSPV-HA1

1.5

1.0

0.5

wtSPV 40

35d

rSPV-HA1

30 20 10

0.0 21d

EMEM

50

Neutralizing antibody titer

IgG against HA1 (OD450)

wtSPV

0

42d

21d

35d

42d

Days post primary immunization

Days post primary immunization

Fig. 3. rSPV-HA1 induced humoral immune response in mice. Serum samples (n = 5) were collected at three time-points. (A) IgG antibodies (OD values) to HA1 were detected with ELISA using a single dilution (1:100). (B) H1N1 SIV-specific neutralizing antibodies were detected by virus neutralizing assay with twofold serial dilution. The titers of neutralizing antibodies were expressed as the reciprocal of the highest serum dilution in which no CPE was observed. Data were shown as mean ± S.D.

5 4

250

B

EMEM wtSPV

EMEM wtSPV

200

rSPV-HA1

Concentration (pg/ml)

Stimulation Index (SI)

A

3 2 1

rSPV-HA1

150 100 50

0 21d

35d

42d

0 IL-4

Days post primary immunization

IFN-Y

Fig. 4. rSPV-HA1 induced cell-mediated immune response. Mouse splenocyte samples (n = 5) were collected at days 21, 35 and 42 dpi and stimulated with purified H1N1 SIV HA1 antigen (10 ␮g/ml) in triplicate. (A) After 45 h stimulation, MTT was added and the proliferation responses were detected by a standard MTT method. Stimulation index is the ratio of OD570nm of stimulated well to that of unstimulated one. The PHA control samples showed a stimulation index of 5–8. (B) After 66 h stimulation, the levels of IFN-␥ and IL-4 in the supernatants. Data are shown as mean ± S.D.

(Fig. 4B). The concentrations of IFN-␥ and IL-4 by the rSPV-HA1 group were significantly higher (P < 0.001, n = 5) than those by the control groups. These data demonstrate that rSPV-HA1 potentiates strong Th1-type and Th2-type cytokine responses.

3.4. Safety of rSPV-HA1 in pigs No pig was found to show any systemic symptoms after vaccination with rSPV-HA1, wtSPV or EMEM. All pig maintained rectal temperature at 38.7–39.3 ◦ C. On day 1 post-vaccination, two pigs (one from the wtSPV and one from the EMEM groups) showed erythemas at the sites of injection. The initial size of the marks was about 3.5 cm in diameter, but it was reduced and disappeared after 3 days. Obviously, the vaccination with rSPV-HA1 and wtSPV was well tolerated by all pigs.

3.6. Protective efficacy of rSPV-HA1 against challenge with H1N1 viruses in pigs During the 5-day observation period, fever and mild respiratory signs including abdominal breathing, sneezing, and nasal discharge were observed only in the control groups (Fig. 6, Table 1). Environmental control pigs (data not shown) and rSPV-HA1 immunized group did not show any clinical signs (Table 1) and maintained steady temperatures (Fig. 6A). No SIV was detected from pigs immunized with rSPV-HA1. However, SIV was found shed from all test pigs inoculated with wtSPV or EMEM (Fig. 6B). Gross lesions in wtSPV and EMEM groups were primarily in the apical and cardiac lung lobes while diaphragmatic and intermediate lobes were 300

EMEM

3.5. rSPV-HA1 induces neutralizing antibody and Th1-type and Th2-type cytokine responses in pigs The pigs vaccinated with rSPV-HA1 had a NA titer 1:17.6 at 21 dpi (Table 1). IL-4 concentrations were 75.91 pg/ml, 72.40 pg/ml and 159.02 pg/ml, and IFN-␥ concentrations were 40.74 pg/ml, 52.81 pg/ml and 247.48 pg/ml in the EMEM, wtSPV or rSPV-HA1 inoculated group, respectively (Fig. 5). The concentrations of IL4 and IFN-␥ in the rSPV-HA1 group were significantly higher (P < 0.01) than those from the control groups. These results suggest that rSPV-HA1 elicits potent Th1-type and Th2-type cytokine responses.

Conce ntration (pg/ml)

wtSPV rSPV-HA1

200

100

0 IL-4

IFN-Y

Fig. 5. Concentration of IL-4 and IFN-␥ in the supernatants of stimulated pig PBMC. The PBMC isolated from the pigs (n = 5) at 21 dpi were stimulated with purified H1N1 SIV virus antigen. After 66 h, the supernatants were collected to examine the levels of IFN-␥ and IL-4. Data are shown as mean ± S.D.

J. Xu et al. / Vaccine 30 (2012) 3119–3125

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Table 1 Neutralizing antibodies and protection results of vaccination after challenge in pigs.a Groups

Pig no.

NA at 21 dpib

Clinical sign scoresc

Gross lesion scoresd

SIV in lungs at 5 dpce

rSPV-HA1

101 102 103 104 105

16 32 8 16 16

0 0 0 0 0

0 0 0 0 0

− − − − −

17.6 ± 8.76A

0A

0A

<4 <4 <4 <4 <4

2.4 1.8 2.2 1.2 0.6

5.7 4.9 5.4 7.1 3.6

<4B

1.6 ± 0.74A

5.3 ± 1.27B

<4 <4 <4 <4 <4

3.0 2.2 1.4 1.6 2.8

7.2 9.2 6.8 4.7 5.4

<4B

2.2 ± 0.71A

6.6 ± 1.75B

Average 201 202 203 204 205

wtSPV

Average 301 301 303 304 305

EMEM

Average

+ + + + +

+ + + + +

a

Within each column, average values followed by different letters (A and B) are significantly different (P < 0.05). The titers of NA were expressed as the reciprocal of the value of the highest serum dilution in which no CPE was observed. Each clinical sign section was given a score of 0 if no symptoms were observed or a score of 1 if a clinical symptom was present. The clinical sign score was calculated as the sum of all clinical sign scores of each pig in 5 days divided by 5 × 2. d Gross lung lesion score was calculated as the sum of percent consolidation of each lung lobe divided by 7. e The positive results were confirmed if one or more samples from one pig were H1N1 SIV positive. b c

less affected. Pigs vaccinated with rSPV-HA1 showed no gross lung pathology (Table 1). More importantly, no H1N1 SIV was isolated from the lungs of the rSPV-HA1 group, but SIV was isolated from all the lungs of the control groups at 5 dpc (Table 1).

Rectal temperature (°C)

A

40.0

EMEM wtSPV rSPV-HA1

39.5

39.0

38.5 0

1

2

3

4

5

Days post challenge

Log TCID 50 /ml

B

Consistently, pigs in the control groups had prominent histopathological changes in the lungs characterized by serious fracture of alveolar septa, shed epithelia, and massive infiltration of lymphocytes and macrophages into the lumen of pulmonary alveoli and small bronchi (Fig. 7b and c). In contrast, the lungs from the rSPV-HA1 group revealed no such changes (Fig. 7a). All the results indicate that rSPV-HA1 inoculation did provide a complete protection against H1N1 SIV challenge in pigs.

2.5

EMEM wtSPV

2.0

rSPV-HA1

1.5 1.0 0.5 0.0 0 0

1

2

3

4

5

Days post challenge Fig. 6. Rectal temperature and nasal shedding patterns post H1N1 SIV challenge. (A) Rectal temperatures of the test pigs (n = 5) post H1N1 SIV challenge. Data are shown as mean ± S.D. (B) Nasal shedding patterns post H1N1 SIV challenge. Data are shown as mean virus titer ± S.D. All test pigs were negative for SIV on the day of challenge.

4. Discussion In the present study we have engineered the swinepox virus to express the HA1 from H1N1 SIV. Such a recombinant virus expresses the HA1 antigen efficiently, induces neutralizing antibodies against H1N1 SIV, potentiates strong Th1-type and Th2-type cytokine responses in mice and pigs, and demonstrates excellent safety and protection efficacy against the virulent homologous SIV challenge in pigs. Mechanistically, rSPV-HA1 induces humoral and cellular immune responses which may both contribute to the protective immunity. The role of HA antibodies against SIV has been reported in previous studies [26–28]. Furthermore, cellmediated immunity has also been reported to confer protective immunity [27,29–31]. Our current study demonstrates that the pigs vaccinated with rSPV-HA1 are well protected from the challenge although the neutralized antibody titers range only from 1:8 to 1:32. This result confirms the role of rSPV-HA1-induced cellular immunity in protection. IFN-␥ is a major immune-modulator for antiviral immunity [32–36]. We have shown that the mice and pigs immunized with rSPV-HA1 produce not only significantly high levels of IL-4 but also high levels of IFN-␥. All these data indicate that rSPV-HA1 could potentiate both Th1-type and Th2-type mediated immunity. In line with our data, several other studies demonstrate that poxvirus vector-based vaccines are capable of inducing potent humoral and T-cell mediated immune responses [6]. The use of poxvirus as a vaccine delivery vector has been extensively explored in mammalian and avian species [6,23,37]. Recombinant fowlpox virus vector has been used as a commercial poultry vaccine against avian influenza virus [6,23], and the

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Fig. 7. Histopathological examination of pig lungs in rSPV-HA1 (a), wtSPV (b) and EMEM group (c) at 5 dpc. Hematoxylin and eosin staining (HE). Magnification, 200×.

recombinant vaccinia rabies vaccine has also been used to successfully eliminate rabies in Western Europe and the United States [38,39]. However, swinepox virus recombinant with SIV as a vaccine vehicle has not been developed. Our study demonstrates that rSPV-HA1 is highly immunogenic, and highly effective in protection of pigs from virulent homologous SIV. Moreover, rSPV-HA1 can be produced in PK15 cells, which makes mass production possible as compared to the traditional SI vaccine production by SPF chicken eggs. It is worthy to point out that SPVs cause considerably slower CPE than those for Orthopoxviruses, such as vaccinia virus [40–45]. This unique feature favors more expression of the engineered-in antigen genes [42,43], which will be highly beneficial for vaccination. The potential value of SPV as a live vector for expressing viral or bacterial genes has been reported [15,17,46]. Due to its biological and clinical safety, its ability to effectively express foreign genes, large packaging capacity for recombinant DNA, easy and low cost of delivery, we predict that SPV could be developed into an excellent vaccine vector for human and animals [16,46]. Although the results of this study are very promising with regard to the vaccines’ immunogenicity, safety and efficacy, it must be taken into account whether there will be the chances and risks of recombination between rSPV-HA1 and wtSPV. Therefore, we examined 2745 pig sera samples with micro-neutralization assay, and no pig was found to be seropositive against SPV (data not shown). Moreover, SPV infections have seldom been found for many years in China. So, we think that the chances of recombination between rSPV-HA1 and wtSPV are rare. In addition, the vaccine is going to be used as attenuated live vaccine as many other live vaccines. It is designed to produce long-term immunity for the vaccinated animal. The vaccine is not developed for multiple usages and therefore immunity against the vector itself is not a major concern for this vaccine. In summary, this study provides the first report to use SPV as a delivery vector for expression of HA1 of H1N1 SIV. Our data indicate that rSPV-HA1 is a promising vaccine candidate for induction of HA1-specific antibodies as well as HA1-specific T-cells. Administration of rSPV-HA1 provides a complete protection against virulent homologous H1N1 SIV challenge in pigs. Thus, this recombinant vaccine is attractive to prevent and control H1N1 SIV in swine.

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Acknowledgment This work was supported by the Special Fund for Public Welfare Industry of the Chinese Ministry of Agriculture (200803016).

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