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ARTICLE IN PRESS
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Vaccine xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Protective efficacy of intranasally administered bivalent live influenza vaccine and immunological mechanisms underlying the protection
1
2
3 4
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Hyun-Mi Pyo, Yan Zhou ∗ Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, SK S7N 5E3, Canada
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6 17
a r t i c l e
i n f o
a b s t r a c t
7 8 9 10 11 12
Article history: Received 11 December 2013 Received in revised form 4 March 2014 Accepted 21 April 2014 Available online xxx
13
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Keywords: Swine influenza virus Live attenuated vaccine
18
1. Introduction
14 15
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Previously we reported the generation of a new potential live attenuated influenza vaccine (LAIV) named SIV/606 that expresses H1 and H3 HAs. We also demonstrated intratracheal vaccination of SIV/606 conferred protection against infections with both H1 and H3 swine influenza virus subtypes in pigs. Here we vaccinated pigs with SIV/606 intranasally, which is a more suitable route for LAIV, and evaluated vaccine efficacy. Intranasal vaccination of SIV/606 induced serum IgG antibody responses against both H1N1 and H3N2 SIVs and high titer of virus neutralizing antibodies against H1N1 SIV but not against H3N2 SIV. When we challenged the pigs with H1N1 and H3N2 SIVs, we observed marked reduction of lung lesions and viral titer in lung tissue in vaccinated pigs. Our analyses also showed that vaccinated pigs had more IFN-␥ secreting cells in trachea–bronchial lymph nodes. Our studies demonstrated that intranasal vaccination of SIV/606 is efficacious for H1N1 and H3N2 SIVs infections. Moreover, our results may help explaining the protection from H3N2 SIV infection despite the low viral neutralizing antibody titer. © 2014 Elsevier Ltd. All rights reserved.
Swine influenza is one of the major respiratory diseases in pigs with high morbidity and low mortality [1]. The significance of endemic swine influenza in pig herd is not only limited to economic loss of pork industry but also to public health. Influenza pandemic in 2009 well demonstrated the risk of interspecies transmission of influenza A virus (IAV). In addition, it also emphasized the role of swine as an intermediate host in interspecies transmission of IAVs [2,3]. As pigs have frequent contact with human, there is always concern of transmission of IAV from swine to humans [4]. Most recently, transmission of swine origin variants H3N2 (H3N2v) and H1N2 (H1N2v) to human was reported [5–7]. These variants are reassortant viruses containing the segment(s) from the 2009 pandemic H1N1 [8,9]. Sero-surveillance study predicted that individuals belonging to certain age groups lacked sero-protection against H3N2v [10]. In addition, the current trivalent human vaccine failed to provide protection against H3N2v infection in a ferret study [11]. Such a continuous emergence
∗ Corresponding author at: Vaccine and Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Road, Saskatoon, SK S7N 5E3, Canada. Tel.: +1 306 966 7716; fax: +1 306 966 7478. E-mail address: [email protected] (Y. Zhou).
of SIVs and the deficiency of protective immunity in human underscore the importance of SIV control in pigs as a preventative measure to stop the swine influenza transmission to human. Vaccination is considered as the most effective countermeasure against influenza infection [12]. However, currently available swine vaccines are inactivated vaccine and the protection is limited to antigenically related virus [13–15]. Compared to inactivated influenza vaccine, LAIVs effectively induce cell-mediated immune (CMI) responses and confer the protection against heterologous or heterosubtypic influenza virus infections [16–18]. Recently, we reported the generation, characterization, immunogenicity and protection efficacy study of a novel recombinant SIV named SIV/606 which contains eight-segment genomic RNA and expresses H1 and H3 hemagglutinins (HAs) [19]. SIV/606 was generated by fusing the H3 HA ectodomain from A/Swine/Texas/4199-2/98 (H3N2) to the cytoplasmic tail, transmembrane domain, and stalk region of neuraminidase (NA) from A/Swine/Saskatchewan/18789/02 (H1N1). SIV/606 was attenuated in pigs and intratracheal vaccination of SIV/606 resulted in promising protection from H1N1 and H3N2 SIVs infections in pigs [19]. Since intratracheal vaccination was not suitable for herd vaccination and is not an ultimate vaccination route, here we investigated the immune responses and protective efficacy after intranasal vaccination of SIV/606 in pigs.
http://dx.doi.org/10.1016/j.vaccine.2014.04.065 0264-410X/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Pyo H-M, Zhou Y. Protective efficacy of intranasally administered bivalent live influenza vaccine and immunological mechanisms underlying the protection. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.04.065
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2. Materials and methods
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2.1. Cells and viruses
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Madin-Darby canine kidney (MDCK) cells were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). SIV/606 was propagated as described previously [19]. The wild type challenged viruses used in this study are A/Swine/Saskatchewan/18789/02 (SIV/SK02) and A/Swine/Texas/4199-2/98 (SIV/TX98) [20,21]. Propagation and titration of these viruses were described in our previous study [22].
70
2.2. Vaccination and viral challenge
63 64 65 66 67 68
71 72 73 74 75 76 77 78 79 80 81 82
Twenty-seven 4-week-old SIV sero-negative pigs were purchased from Prairie Swine Centre Inc. (Saskatoon, Canada) and randomly divided into five groups. Each group had six pigs except group 5, which had three pigs serving as no treatment controls (Table 1). After one week acclimatization, pigs in groups 1 and 3 were mock vaccinated intranasally with 1 ml of MEM (0.5 ml/nostril); whereas pigs in groups 2 and 4 were vaccinated intranasally with 1 ml of MEM containing 1 × 107 PFU of SIV/606 (0.5 ml/nostril; day 0). Pigs were vaccinated twice at three-week interval, and then challenged intratracheally with 8 × 105 PFU of either SIV/SK02 (groups 1 and 2) or SIV/TX98 (groups 3 and 4). Animal trial and experimental procedures performed at the Vaccine
Table 1 Assignment of pigs for nasal vaccination and viral challenge. Group
1 (n = 6) 2 (n = 6) 3 (n = 6) 4 (n = 6) 5 (n = 3)
Vaccination (IN)
Challenge (IT) (day 31)
1 (day 0)
2 (day 21)
MEM SIV/606 MEM SIV/606 –
MEM SIV/606 MEM SIV/606 –
SIV/SK02 SIV/SK02 SIV/TX98 SIV/TX98 –
and Infectious Disease Organization, were complying with the ethical guidelines of the University of Saskatchewan and the Canadian Council of Animal Care. 2.3. Sample collection Serum samples were collected on days 0, 21, 31, and 36. After viral challenge on day 31, pigs were monitored daily for body temperature and clinical signs of SIV infection for five days. On day 36, all pigs were euthanized and lung lesions corresponding to SIV infection were examined and recorded. Lung tissue punches were collected from right cranial, middle and caudal lobes using 8 mm biopsy punch (Miltex, Inc., York, PA, USA) for viral isolation and histopathological analysis. Broncho-alveolar fluid (BALF) was collected by rinsing the left cranial, middle and caudal lobes with 50 ml
Fig. 1. Antigen specific serum IgG response in pigs prior to and post the SIV/606 intranasal vaccination. Serum IgG specific to SIV/SK02 (A) and SIV/TX98 (B) were detected from the serum collected before the vaccination (day 0) and after the first and second vaccinations of SIV/606 (day 21 and 31). Each dot represents readout from individual animals. Mean value of each group is indicated by a horizontal bar (*p < 0.05; ****p < 0.0001).
Please cite this article in press as: Pyo H-M, Zhou Y. Protective efficacy of intranasally administered bivalent live influenza vaccine and immunological mechanisms underlying the protection. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.04.065
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Fig. 2. Virus neutralizing antibody titers against SIV/SK02 (A) and SIV/TX98 (B) prior to and post the virus challenge. Serum samples collected on day 31 and day 36 were subjected to virus neutralization assay. Each dot represents readout from individual animals. Bars indicate mean values of each group (**p < 0.01; ***p < 0.001).
2.7. Lymphoproliferation (LPR) assay
99
MEM, and was used for measuring IgA. Trachea–bronchial lymph nodes (LNs) were collected in AIM-V medium supplemented with 1% penicillin–streptomycin (Invitrogen, Canada). Lymph node cells (LNCs) were prepared as previously described [23].
100
2.4. Enzyme-linked immunosorbent assay (ELISA)
2.8. Porcine IFN- enzyme-linked immunospot (ELISPOT) assay
IDEXX SIV H1N1 Ab test and IDEXX SIV H3N2 Ab test (IDEXX Laboratories, Inc., Westbrook, Maine, USA) were used to screen the sows and piglets. Antigen specific serum IgG and BALF IgA were measured as previously described [23].
Porcine IFN-␥ secreting LNCs were measured as previously described [23].
96 97 98
101 102 103 104
LNC proliferation upon antigen stimulation was measured by the uptake of 3 H as previously described [23].
2.9. Histopathology 105
106 107
108
109 110 111 112 113 114 115 116
2.5. Virus neutralization assay Virus neutralization assay was performed as described in “WHO manual on animal influenza diagnosis and surveillance” [24].
Tissue sections of lungs were stained with hematoxylin and eosin, and examined for the bronchiolar epithelial changes and peribronchiolar inflammation by a single pathologist under the blind condition for the experimental groups as described previously [20]. Histopathology of lung tissue was scored scale 0 (no visible change) to 4 (severe diffuse change).
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121 122
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124 125 126 127 128 129
2.6. Virus isolation and titration Virus titers in lung tissues were determined in accordance with “The processing clinical material for virus isolation” described in “WHO manual on animal diagnosis and surveillance” [24]. Lung tissues were ground using conical tissue grinder (VWR International) in MEM. The supernatant after the centrifugation of tissue ground was serially diluted and then was inoculated onto MDCK cells grown in 96-well flat bottom culture plate. Titers were calculated in TCID50 according to the Reed–Muench method [25].
2.10. Statistics Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, Inc., USA). One-way ANOVA followed by Tukey’s multiple comparison was used to determine the statistical significance of the difference between the groups. For lung viral titers and LPR results were transformed by rank for statistical analysis.
Please cite this article in press as: Pyo H-M, Zhou Y. Protective efficacy of intranasally administered bivalent live influenza vaccine and immunological mechanisms underlying the protection. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.04.065
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Fig. 3. Record of body temperature in 5 days post virus challenge with SIV/SK02 (A) and SIV/TX98 (B). Daily body temperature was presented by mean ± standard deviation.
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3. Results 3.1. Serum IgG and virus neutralizing titers after intranasal vaccination of SIV/606 All piglets from SIV sero-negative sows were H1N1 and H3N2 SIVs negative when examined using commercial diagnostic ELISA (data not shown). All piglets were sero-negative in antigen specific ELISA against SIV/SK02 and SIV/TX98 on day 0. Following the first intranasal vaccination of SIV/606 (on day 21), 5 out of 12 pigs in two of the vaccine groups developed SIV/SK02 and SIV/TX98 specific serum IgG. Four out of 12 pigs generated either SIV/SK02 or SIV/TX98 specific serum IgG. Following the second vaccination (on day 31), all vaccinated pigs showed serum IgG against both SIV/SK02 and SIV/TX98 (Fig. 1A and B). It was noted that intranasal vaccination of SIV/606 induced higher IgG against SIV/SK02 than SIV/TX98. Virus neutralization assay using serum collected before virus challenge (on day 31) showed all SIV/606 vaccinated pigs had neutralizing antibodies to SIV/SK02 (Fig. 2A, mean = 145) while 8 pigs out of 12 had neutralizing antibodies to SIV/TX98 (Fig. 2B, mean = 35). We did not detect virus neutralizing antibody to SIV/SK02 or SIV/TX98 in pigs belonging to groups 1, 3 and 5. 3.2. Intranasal vaccination of SIV/606 protected pigs from H1N1 and H3N2 SIVs infections. On day 31, pigs in groups 1–4 were challenged with either SIV/SK02 or SIV/TX98 and observed for five days. During the observation, there were no obvious clinical signs in all pigs except for fever. Pigs challenged with SIV/SK02 had elevated body temperature on day 1 post challenge, however, there was no difference
between vaccinated group (40.9 ± 0.4 ◦ C) and unvaccinated group (40.9 ± 0.3 ◦ C) (Fig. 3A). Pigs challenged with SIV/TX98 showed mild fever on day 1 post challenge. Even though the body temperature of pigs vaccinated with SIV/606 (40.0 ± 0.4 ◦ C) was slightly lower than unvaccinated group pigs (40.4 ± 0.4 ◦ C), the difference was not statistically significant (Fig. 3B). We did not observe such body temperature fluctuation in environmental control pigs (group 5, 39.5 ± 0.3 ◦ C). Necropsy was performed on day 5 post viral challenge and lungs were assessed for the presence of SIV infection induced gross lesions, characterized by distinctive demarcation of dark red colored consolidation. Pigs in unvaccinated and challenged groups, developed moderate to severe lesions in the lung, with the mean score of 8.00 and 7.65, respectively (Fig. 4A). In contrast, SIV/606 vaccinated and virus challenged groups showed less gross lesions. Specifically, 4 out of 6 pigs in SIV/606 vaccinated and SIV/SK02 challenged group had no gross lesions in their lungs and the remaining 2 pigs developed lesions in small areas (mean score of 0.91). Two pigs in SIV/606 vaccinated and SIV/TX98 challenged group had no lesions and 4 pigs had mild gross lesions (mean score of 2.63). Pigs in environmental control group had no SIV induced gross lesions (mean score of 0.33). SIV infection caused histopathological changes in lung tissue such as necrosis of bronchiolar epithelial cells and neutrophil infiltration in interstitial and peribronchial areas. SIV/606 vaccinated and virus challenged groups developed less severe histopathology than their corresponding unvaccinated and challenged groups (Fig. 4B). Pigs in unvaccinated and SIV/SK02 challenged group developed mild to moderate bronchointerstitial pneumonia (mean histopathology score of 1.8). Pigs in SIV/606 vaccinated and SIV/SK02 challenged group, developed mild interstitial pneumonia (mean histopathology score of 1.3). Pigs in unvaccinated and
Please cite this article in press as: Pyo H-M, Zhou Y. Protective efficacy of intranasally administered bivalent live influenza vaccine and immunological mechanisms underlying the protection. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.04.065
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Fig. 4. Gross lesion score (A), histopathological score (B), and virus titer (C) in lung tissue post virus challenge. (A) At necropsy, SIV induced gross lesion in lung was examined and recorded by percentage of its portion in each lobe. Then, contribution ratio of each lobe to total lung volume was reflected to the percentage and the sum of adjusted percentage values presented as a final score for individual pig. (B) Lung tissues were collected and processed for histopathological examination. Lesion severity was scored according to the distribution of microscopic lesions within the sections examined, from scale 0 to 4; 0, no visible changes; 1, mild focal or multifocal change; 2, moderate multifocal change; 3, moderate diffuse change; 4, severe diffuse change. N = 5 in group 3 due to the loss of one tissue section. (C) Lung tissues were homogenized, and virus titers were determined by plaque assay on MDCK cells. Each dot represents readout from individual animals. Bars represent mean values of each group (*p < 0.05; **p < 0.01; ****p < 0.0001).
197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221
SIV/TX98 challenged group developed the most severe histopathological lesions (mean histopathology score of 2.6) which were characterized by moderate bronchointerstitial pneumonia with necrosis of bronchiolar epithelial cells and infiltration of inflammatory cells. Pigs in SIV/606 vaccinated and SIV/TX98 challenged group, had mild histopathology which was characterized as moderate bronchointerstitial pneumonia (mean histopathology score of 2). The low score in histopathology of SIV/606 vaccinated pigs is mainly due to the reduction of inflammatory cell in lung tissues. Compared to their unvaccinated counter control pigs, SIV/606 vaccinated pigs showed less neutrophil infiltration (data not shown). Viruses were isolated from lungs of 5 out of 6 pigs in both groups 1 and 3, which were unvaccinated and challenged with SIV/SK02 and SIV/TX98 (Fig. 4C). The mean viral titers in groups 1 and 3 were 104.39 and 104.87 TCID50 /g, respectively. One pig from each group 1 and 3 had no virus detected in lung tissue samples. Among these two pigs, a pig from group 3 was the animal that developed a mild lung gross lesion (gross lesion score of 1.25) and a low histopathological lesion (histopathology score of 1). The other pig from group 1 developed a severe gross lesion and histopathology (gross lesion score of 10.75 and histopathology score of 3). No virus was recovered from any of the pigs in groups 2 and 4, SIV/606 vaccinated and challenged, as well as the environmental control pigs in group 5.
3.3. Post challenge immune responses Antigen specific LPR and IFN-␥ ELISPOT were performed to evaluate the CMI. The LNCs were isolated from tracheo-bronchial LNs thus the results reflect local CMI responses developed following vaccination and viral challenge. LNCs from virus challenged pigs in groups 1–4 proliferated when they were stimulated in vitro with respective antigens (Fig. 5A and B), regardless of being vaccinated or not. SIV/606 vaccinated pigs in groups 2 and 4 showed slightly higher proliferation compared to unvaccinated pigs in groups 1 and 3, although the difference was not statistically significant. IFN-␥ ELISPOT results showed that pigs vaccinated with SIV/606 had significantly more antigen-specific IFN-␥ secreting cells than unvaccinated pigs (Fig. 5C and D). Pigs in group 2, SIV/606 vaccinated and SIV/SK02 challenged, had more SIV/SK02 specific IFN-␥ producing cells (mean value is 452) than pigs in group 1 (mean value of 202) (Fig. 5C). Similarly, a significantly high number of IFN-␥ secreting cells were detected in group 4 pigs (mean value of 634) than that in group 3 pigs (mean value of 159). LNCs from the pigs in group 5 did not respond to viral antigen stimulation in either LPR or IFN-␥ ELISPOT assay. Serum and BALF samples collected at necropsy were subjected to antigen specific IgG and IgA ELISA (Fig. 6) and viral neutralization assay (Fig. 2). No serum IgG or BALF IgA specific to either SIV/SK02 or SIV/TX98 was detected from pigs in group 5. In both vaccinated
Please cite this article in press as: Pyo H-M, Zhou Y. Protective efficacy of intranasally administered bivalent live influenza vaccine and immunological mechanisms underlying the protection. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.04.065
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B
A
p=0.0093
*
Stimulationindex
Stimulationindex
p=0.0139
500 p=0.0374
*
400 300 200 100
500 p=0.0424
**
*
400 300 200 100 0
0 Vaccination:
MEM
Challenge:
-
MEM
SIV/606 SIV/SK02
Vaccination:
MEM
Challenge:
-
MEM
SIV/606 SIV/TX98
D
C
NumberofIFN- secreting cells 6 (per 10 cells)
Number ofIFN- secreting cells 6 (per 10 cells)
p
ARTICLE IN PRESS
JVAC 15346 1–8
Vaccine xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Protective efficacy of intranasally administered bivalent live influenza vaccine and immunological mechanisms underlying the protection
1
2
3 4
Q1
Hyun-Mi Pyo, Yan Zhou ∗ Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, SK S7N 5E3, Canada
5
6 17
a r t i c l e
i n f o
a b s t r a c t
7 8 9 10 11 12
Article history: Received 11 December 2013 Received in revised form 4 March 2014 Accepted 21 April 2014 Available online xxx
13
16
Keywords: Swine influenza virus Live attenuated vaccine
18
1. Introduction
14 15
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Previously we reported the generation of a new potential live attenuated influenza vaccine (LAIV) named SIV/606 that expresses H1 and H3 HAs. We also demonstrated intratracheal vaccination of SIV/606 conferred protection against infections with both H1 and H3 swine influenza virus subtypes in pigs. Here we vaccinated pigs with SIV/606 intranasally, which is a more suitable route for LAIV, and evaluated vaccine efficacy. Intranasal vaccination of SIV/606 induced serum IgG antibody responses against both H1N1 and H3N2 SIVs and high titer of virus neutralizing antibodies against H1N1 SIV but not against H3N2 SIV. When we challenged the pigs with H1N1 and H3N2 SIVs, we observed marked reduction of lung lesions and viral titer in lung tissue in vaccinated pigs. Our analyses also showed that vaccinated pigs had more IFN-␥ secreting cells in trachea–bronchial lymph nodes. Our studies demonstrated that intranasal vaccination of SIV/606 is efficacious for H1N1 and H3N2 SIVs infections. Moreover, our results may help explaining the protection from H3N2 SIV infection despite the low viral neutralizing antibody titer. © 2014 Elsevier Ltd. All rights reserved.
Swine influenza is one of the major respiratory diseases in pigs with high morbidity and low mortality [1]. The significance of endemic swine influenza in pig herd is not only limited to economic loss of pork industry but also to public health. Influenza pandemic in 2009 well demonstrated the risk of interspecies transmission of influenza A virus (IAV). In addition, it also emphasized the role of swine as an intermediate host in interspecies transmission of IAVs [2,3]. As pigs have frequent contact with human, there is always concern of transmission of IAV from swine to humans [4]. Most recently, transmission of swine origin variants H3N2 (H3N2v) and H1N2 (H1N2v) to human was reported [5–7]. These variants are reassortant viruses containing the segment(s) from the 2009 pandemic H1N1 [8,9]. Sero-surveillance study predicted that individuals belonging to certain age groups lacked sero-protection against H3N2v [10]. In addition, the current trivalent human vaccine failed to provide protection against H3N2v infection in a ferret study [11]. Such a continuous emergence
∗ Corresponding author at: Vaccine and Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Road, Saskatoon, SK S7N 5E3, Canada. Tel.: +1 306 966 7716; fax: +1 306 966 7478. E-mail address: [email protected] (Y. Zhou).
of SIVs and the deficiency of protective immunity in human underscore the importance of SIV control in pigs as a preventative measure to stop the swine influenza transmission to human. Vaccination is considered as the most effective countermeasure against influenza infection [12]. However, currently available swine vaccines are inactivated vaccine and the protection is limited to antigenically related virus [13–15]. Compared to inactivated influenza vaccine, LAIVs effectively induce cell-mediated immune (CMI) responses and confer the protection against heterologous or heterosubtypic influenza virus infections [16–18]. Recently, we reported the generation, characterization, immunogenicity and protection efficacy study of a novel recombinant SIV named SIV/606 which contains eight-segment genomic RNA and expresses H1 and H3 hemagglutinins (HAs) [19]. SIV/606 was generated by fusing the H3 HA ectodomain from A/Swine/Texas/4199-2/98 (H3N2) to the cytoplasmic tail, transmembrane domain, and stalk region of neuraminidase (NA) from A/Swine/Saskatchewan/18789/02 (H1N1). SIV/606 was attenuated in pigs and intratracheal vaccination of SIV/606 resulted in promising protection from H1N1 and H3N2 SIVs infections in pigs [19]. Since intratracheal vaccination was not suitable for herd vaccination and is not an ultimate vaccination route, here we investigated the immune responses and protective efficacy after intranasal vaccination of SIV/606 in pigs.
http://dx.doi.org/10.1016/j.vaccine.2014.04.065 0264-410X/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Pyo H-M, Zhou Y. Protective efficacy of intranasally administered bivalent live influenza vaccine and immunological mechanisms underlying the protection. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.04.065
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2. Materials and methods
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2.1. Cells and viruses
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Madin-Darby canine kidney (MDCK) cells were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). SIV/606 was propagated as described previously [19]. The wild type challenged viruses used in this study are A/Swine/Saskatchewan/18789/02 (SIV/SK02) and A/Swine/Texas/4199-2/98 (SIV/TX98) [20,21]. Propagation and titration of these viruses were described in our previous study [22].
70
2.2. Vaccination and viral challenge
63 64 65 66 67 68
71 72 73 74 75 76 77 78 79 80 81 82
Twenty-seven 4-week-old SIV sero-negative pigs were purchased from Prairie Swine Centre Inc. (Saskatoon, Canada) and randomly divided into five groups. Each group had six pigs except group 5, which had three pigs serving as no treatment controls (Table 1). After one week acclimatization, pigs in groups 1 and 3 were mock vaccinated intranasally with 1 ml of MEM (0.5 ml/nostril); whereas pigs in groups 2 and 4 were vaccinated intranasally with 1 ml of MEM containing 1 × 107 PFU of SIV/606 (0.5 ml/nostril; day 0). Pigs were vaccinated twice at three-week interval, and then challenged intratracheally with 8 × 105 PFU of either SIV/SK02 (groups 1 and 2) or SIV/TX98 (groups 3 and 4). Animal trial and experimental procedures performed at the Vaccine
Table 1 Assignment of pigs for nasal vaccination and viral challenge. Group
1 (n = 6) 2 (n = 6) 3 (n = 6) 4 (n = 6) 5 (n = 3)
Vaccination (IN)
Challenge (IT) (day 31)
1 (day 0)
2 (day 21)
MEM SIV/606 MEM SIV/606 –
MEM SIV/606 MEM SIV/606 –
SIV/SK02 SIV/SK02 SIV/TX98 SIV/TX98 –
and Infectious Disease Organization, were complying with the ethical guidelines of the University of Saskatchewan and the Canadian Council of Animal Care. 2.3. Sample collection Serum samples were collected on days 0, 21, 31, and 36. After viral challenge on day 31, pigs were monitored daily for body temperature and clinical signs of SIV infection for five days. On day 36, all pigs were euthanized and lung lesions corresponding to SIV infection were examined and recorded. Lung tissue punches were collected from right cranial, middle and caudal lobes using 8 mm biopsy punch (Miltex, Inc., York, PA, USA) for viral isolation and histopathological analysis. Broncho-alveolar fluid (BALF) was collected by rinsing the left cranial, middle and caudal lobes with 50 ml
Fig. 1. Antigen specific serum IgG response in pigs prior to and post the SIV/606 intranasal vaccination. Serum IgG specific to SIV/SK02 (A) and SIV/TX98 (B) were detected from the serum collected before the vaccination (day 0) and after the first and second vaccinations of SIV/606 (day 21 and 31). Each dot represents readout from individual animals. Mean value of each group is indicated by a horizontal bar (*p < 0.05; ****p < 0.0001).
Please cite this article in press as: Pyo H-M, Zhou Y. Protective efficacy of intranasally administered bivalent live influenza vaccine and immunological mechanisms underlying the protection. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.04.065
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Fig. 2. Virus neutralizing antibody titers against SIV/SK02 (A) and SIV/TX98 (B) prior to and post the virus challenge. Serum samples collected on day 31 and day 36 were subjected to virus neutralization assay. Each dot represents readout from individual animals. Bars indicate mean values of each group (**p < 0.01; ***p < 0.001).
2.7. Lymphoproliferation (LPR) assay
99
MEM, and was used for measuring IgA. Trachea–bronchial lymph nodes (LNs) were collected in AIM-V medium supplemented with 1% penicillin–streptomycin (Invitrogen, Canada). Lymph node cells (LNCs) were prepared as previously described [23].
100
2.4. Enzyme-linked immunosorbent assay (ELISA)
2.8. Porcine IFN- enzyme-linked immunospot (ELISPOT) assay
IDEXX SIV H1N1 Ab test and IDEXX SIV H3N2 Ab test (IDEXX Laboratories, Inc., Westbrook, Maine, USA) were used to screen the sows and piglets. Antigen specific serum IgG and BALF IgA were measured as previously described [23].
Porcine IFN-␥ secreting LNCs were measured as previously described [23].
96 97 98
101 102 103 104
LNC proliferation upon antigen stimulation was measured by the uptake of 3 H as previously described [23].
2.9. Histopathology 105
106 107
108
109 110 111 112 113 114 115 116
2.5. Virus neutralization assay Virus neutralization assay was performed as described in “WHO manual on animal influenza diagnosis and surveillance” [24].
Tissue sections of lungs were stained with hematoxylin and eosin, and examined for the bronchiolar epithelial changes and peribronchiolar inflammation by a single pathologist under the blind condition for the experimental groups as described previously [20]. Histopathology of lung tissue was scored scale 0 (no visible change) to 4 (severe diffuse change).
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2.6. Virus isolation and titration Virus titers in lung tissues were determined in accordance with “The processing clinical material for virus isolation” described in “WHO manual on animal diagnosis and surveillance” [24]. Lung tissues were ground using conical tissue grinder (VWR International) in MEM. The supernatant after the centrifugation of tissue ground was serially diluted and then was inoculated onto MDCK cells grown in 96-well flat bottom culture plate. Titers were calculated in TCID50 according to the Reed–Muench method [25].
2.10. Statistics Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, Inc., USA). One-way ANOVA followed by Tukey’s multiple comparison was used to determine the statistical significance of the difference between the groups. For lung viral titers and LPR results were transformed by rank for statistical analysis.
Please cite this article in press as: Pyo H-M, Zhou Y. Protective efficacy of intranasally administered bivalent live influenza vaccine and immunological mechanisms underlying the protection. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.04.065
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Fig. 3. Record of body temperature in 5 days post virus challenge with SIV/SK02 (A) and SIV/TX98 (B). Daily body temperature was presented by mean ± standard deviation.
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3. Results 3.1. Serum IgG and virus neutralizing titers after intranasal vaccination of SIV/606 All piglets from SIV sero-negative sows were H1N1 and H3N2 SIVs negative when examined using commercial diagnostic ELISA (data not shown). All piglets were sero-negative in antigen specific ELISA against SIV/SK02 and SIV/TX98 on day 0. Following the first intranasal vaccination of SIV/606 (on day 21), 5 out of 12 pigs in two of the vaccine groups developed SIV/SK02 and SIV/TX98 specific serum IgG. Four out of 12 pigs generated either SIV/SK02 or SIV/TX98 specific serum IgG. Following the second vaccination (on day 31), all vaccinated pigs showed serum IgG against both SIV/SK02 and SIV/TX98 (Fig. 1A and B). It was noted that intranasal vaccination of SIV/606 induced higher IgG against SIV/SK02 than SIV/TX98. Virus neutralization assay using serum collected before virus challenge (on day 31) showed all SIV/606 vaccinated pigs had neutralizing antibodies to SIV/SK02 (Fig. 2A, mean = 145) while 8 pigs out of 12 had neutralizing antibodies to SIV/TX98 (Fig. 2B, mean = 35). We did not detect virus neutralizing antibody to SIV/SK02 or SIV/TX98 in pigs belonging to groups 1, 3 and 5. 3.2. Intranasal vaccination of SIV/606 protected pigs from H1N1 and H3N2 SIVs infections. On day 31, pigs in groups 1–4 were challenged with either SIV/SK02 or SIV/TX98 and observed for five days. During the observation, there were no obvious clinical signs in all pigs except for fever. Pigs challenged with SIV/SK02 had elevated body temperature on day 1 post challenge, however, there was no difference
between vaccinated group (40.9 ± 0.4 ◦ C) and unvaccinated group (40.9 ± 0.3 ◦ C) (Fig. 3A). Pigs challenged with SIV/TX98 showed mild fever on day 1 post challenge. Even though the body temperature of pigs vaccinated with SIV/606 (40.0 ± 0.4 ◦ C) was slightly lower than unvaccinated group pigs (40.4 ± 0.4 ◦ C), the difference was not statistically significant (Fig. 3B). We did not observe such body temperature fluctuation in environmental control pigs (group 5, 39.5 ± 0.3 ◦ C). Necropsy was performed on day 5 post viral challenge and lungs were assessed for the presence of SIV infection induced gross lesions, characterized by distinctive demarcation of dark red colored consolidation. Pigs in unvaccinated and challenged groups, developed moderate to severe lesions in the lung, with the mean score of 8.00 and 7.65, respectively (Fig. 4A). In contrast, SIV/606 vaccinated and virus challenged groups showed less gross lesions. Specifically, 4 out of 6 pigs in SIV/606 vaccinated and SIV/SK02 challenged group had no gross lesions in their lungs and the remaining 2 pigs developed lesions in small areas (mean score of 0.91). Two pigs in SIV/606 vaccinated and SIV/TX98 challenged group had no lesions and 4 pigs had mild gross lesions (mean score of 2.63). Pigs in environmental control group had no SIV induced gross lesions (mean score of 0.33). SIV infection caused histopathological changes in lung tissue such as necrosis of bronchiolar epithelial cells and neutrophil infiltration in interstitial and peribronchial areas. SIV/606 vaccinated and virus challenged groups developed less severe histopathology than their corresponding unvaccinated and challenged groups (Fig. 4B). Pigs in unvaccinated and SIV/SK02 challenged group developed mild to moderate bronchointerstitial pneumonia (mean histopathology score of 1.8). Pigs in SIV/606 vaccinated and SIV/SK02 challenged group, developed mild interstitial pneumonia (mean histopathology score of 1.3). Pigs in unvaccinated and
Please cite this article in press as: Pyo H-M, Zhou Y. Protective efficacy of intranasally administered bivalent live influenza vaccine and immunological mechanisms underlying the protection. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.04.065
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Fig. 4. Gross lesion score (A), histopathological score (B), and virus titer (C) in lung tissue post virus challenge. (A) At necropsy, SIV induced gross lesion in lung was examined and recorded by percentage of its portion in each lobe. Then, contribution ratio of each lobe to total lung volume was reflected to the percentage and the sum of adjusted percentage values presented as a final score for individual pig. (B) Lung tissues were collected and processed for histopathological examination. Lesion severity was scored according to the distribution of microscopic lesions within the sections examined, from scale 0 to 4; 0, no visible changes; 1, mild focal or multifocal change; 2, moderate multifocal change; 3, moderate diffuse change; 4, severe diffuse change. N = 5 in group 3 due to the loss of one tissue section. (C) Lung tissues were homogenized, and virus titers were determined by plaque assay on MDCK cells. Each dot represents readout from individual animals. Bars represent mean values of each group (*p < 0.05; **p < 0.01; ****p < 0.0001).
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SIV/TX98 challenged group developed the most severe histopathological lesions (mean histopathology score of 2.6) which were characterized by moderate bronchointerstitial pneumonia with necrosis of bronchiolar epithelial cells and infiltration of inflammatory cells. Pigs in SIV/606 vaccinated and SIV/TX98 challenged group, had mild histopathology which was characterized as moderate bronchointerstitial pneumonia (mean histopathology score of 2). The low score in histopathology of SIV/606 vaccinated pigs is mainly due to the reduction of inflammatory cell in lung tissues. Compared to their unvaccinated counter control pigs, SIV/606 vaccinated pigs showed less neutrophil infiltration (data not shown). Viruses were isolated from lungs of 5 out of 6 pigs in both groups 1 and 3, which were unvaccinated and challenged with SIV/SK02 and SIV/TX98 (Fig. 4C). The mean viral titers in groups 1 and 3 were 104.39 and 104.87 TCID50 /g, respectively. One pig from each group 1 and 3 had no virus detected in lung tissue samples. Among these two pigs, a pig from group 3 was the animal that developed a mild lung gross lesion (gross lesion score of 1.25) and a low histopathological lesion (histopathology score of 1). The other pig from group 1 developed a severe gross lesion and histopathology (gross lesion score of 10.75 and histopathology score of 3). No virus was recovered from any of the pigs in groups 2 and 4, SIV/606 vaccinated and challenged, as well as the environmental control pigs in group 5.
3.3. Post challenge immune responses Antigen specific LPR and IFN-␥ ELISPOT were performed to evaluate the CMI. The LNCs were isolated from tracheo-bronchial LNs thus the results reflect local CMI responses developed following vaccination and viral challenge. LNCs from virus challenged pigs in groups 1–4 proliferated when they were stimulated in vitro with respective antigens (Fig. 5A and B), regardless of being vaccinated or not. SIV/606 vaccinated pigs in groups 2 and 4 showed slightly higher proliferation compared to unvaccinated pigs in groups 1 and 3, although the difference was not statistically significant. IFN-␥ ELISPOT results showed that pigs vaccinated with SIV/606 had significantly more antigen-specific IFN-␥ secreting cells than unvaccinated pigs (Fig. 5C and D). Pigs in group 2, SIV/606 vaccinated and SIV/SK02 challenged, had more SIV/SK02 specific IFN-␥ producing cells (mean value is 452) than pigs in group 1 (mean value of 202) (Fig. 5C). Similarly, a significantly high number of IFN-␥ secreting cells were detected in group 4 pigs (mean value of 634) than that in group 3 pigs (mean value of 159). LNCs from the pigs in group 5 did not respond to viral antigen stimulation in either LPR or IFN-␥ ELISPOT assay. Serum and BALF samples collected at necropsy were subjected to antigen specific IgG and IgA ELISA (Fig. 6) and viral neutralization assay (Fig. 2). No serum IgG or BALF IgA specific to either SIV/SK02 or SIV/TX98 was detected from pigs in group 5. In both vaccinated
Please cite this article in press as: Pyo H-M, Zhou Y. Protective efficacy of intranasally administered bivalent live influenza vaccine and immunological mechanisms underlying the protection. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.04.065
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B
A
p=0.0093
*
Stimulationindex
Stimulationindex
p=0.0139
500 p=0.0374
*
400 300 200 100
500 p=0.0424
**
*
400 300 200 100 0
0 Vaccination:
MEM
Challenge:
-
MEM
SIV/606 SIV/SK02
Vaccination:
MEM
Challenge:
-
MEM
SIV/606 SIV/TX98
D
C
NumberofIFN- secreting cells 6 (per 10 cells)
Number ofIFN- secreting cells 6 (per 10 cells)
p
