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Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens
Vaccine xxx (2018) xxx–xxx
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Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens Jake Astill a, Tamiru Alkie a,b, Alexander Yitbarek a, Khaled Taha Abd Abdelaziz c, Jegarubee Bavananthasivam a, Éva Nagy a, James John Petrik d, Shayan Sharif a,⇑ a
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada Department of Biology, Wilfred Laurier University, Waterloo, ON N2L 3C5, Canada1 c Pathology Department, Faculty of Veterinary Medicine, Beni-Suef University, Al Shamlah, 62511 Beni-Suef, Egypt d Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada b
a r t i c l e
i n f o
Article history: Received 2 March 2018 Received in revised form 20 May 2018 Accepted 21 May 2018 Available online xxxx Keywords: H9N2 AIV Whole inactivated virus vaccine Adaptive immunity Chickens Toll-like receptor 21 CpG ODN 2007
a b s t r a c t Several types of avian influenza virus (AIV) vaccines exist, including live-attenuated, vectored, and whole inactivated virus (WIV) vaccines. Inactivated vaccines offer some advantages compared to other types of vaccines, including ease of production and lack of ability to revert to a virulent state. However, WIV are poorly immunogenic, especially when these vaccines are delivered to mucosal surfaces. There are several factors that contribute to the immunogenicity of vaccines, one of which is the method used to inactivate viruses. Several methods exist for producing influenza WIVs, including formaldehyde, a chemical that affects protein structures leading to virus inactivation. Other methods include treatment with betapropiolactone (BPL) and the application of gamma radiation, both of which have less effects on protein structures compared to formaldehyde, and instead alter nucleic acids in the virion. Here, we sought to determine the effect of the above inactivation methods on immunogenicity of AIV vaccines. To this end, chickens were vaccinated with three different H9N2 WIVs using formaldehyde, BPL, and gamma radiation for inactivation. In addition to administering these three WIVs alone as vaccines, we also included CpG ODN 2007, a synthetic ligand recognized by Toll-like receptor (TLR)21 in chickens, as an adjuvant for each WIV. Subsequently, antibody- and cell-mediated immune responses were measured following vaccination. Antibody-mediated immune responses were increased in chickens that received the BPL and Gamma WIVs compared to the formaldehyde WIV. CpG ODN 2007 was found to significantly increase antibody responses for each WIV compared to WIV alone. Furthermore, we observed the presence of cell-mediated immune responses in chickens that received the BPL WIV combined with CpG ODN 2007. Based on these results, the BPL WIV + CpG ODN 2007 combination was the most effective vaccine at inducing adaptive immune responses against H9N2 AIV. Future studies should characterize mucosal adaptive immune responses to these vaccines. Ó 2018 Elsevier Ltd. All rights reserved.
1. Introduction Low pathogenic avian influenza viruses (LPAIV) are a class of avian influenza viruses (AIV) that commonly infect wild birds without causing any signs of clinical disease [1]. H9N2 AIV is a LPAIV that has become a concern for economical and health related reasons. H9N2 virus infection in chickens can decrease egg produc-
⇑ Corresponding author. 1
E-mail address: [email protected] (S. Sharif). Present address.
tion, and co-infection with other infectious pathogens can result in immunosuppression and pathological changes [2,3]. There is strong evidence of human exposure to the H9N2 virus among poultry workers in certain regions of the world including China and India [4,5], although not many cases of infection are reported. H9N2 AIV is also responsible for contributing its internal genes to a reassortant H7N9 virus which has demonstrated 40% mortality in human cases in China [6]. Vaccination is a potential strategy that can be used to control H9N2 infection in poultry. Many types of influenza vaccines exist, including live-attenuated vaccines, subunit vaccines, and
https://doi.org/10.1016/j.vaccine.2018.05.093 0264-410X/Ó 2018 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Astill J et al. Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens. Vaccine (2018), https://doi.org/10.1016/j. vaccine.2018.05.093
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inactivated vaccines. Whole-inactivated virus (WIV) vaccines for influenza were first explored in the 1940s, when formaldehyde and other compounds were used to inactivate influenza viruses [7]. Since then, many different ways to produce influenza WIV vaccines have emerged, including other chemical treatments or the application of different forms of electromagnetic radiation [8]. Two common chemicals used to produce influenza WIVs include formaldehyde and beta-propiolactone (BPL). As an aldehyde, formaldehyde reacts with protein structures, crosslinking various amino acids. This in turn affects the function of proteins and can lead to a loss of virus infectivity [9]. Altering protein structures is an effective way to induce virus inactivation, however, this can change the outcome of the elicited immune response following vaccination due to changes on epitopes of immunogenic proteins, such as the influenza hemagglutinin (HA) protein [9]. BPL is an electrophilic compound that reacts with nucleic acids, mainly with the nucleotides guanosine and adenosine [8]. These reactions alter the structure of nucleic acids inducing changes such as strand breaks and improper linkages between nucleic acids and protein, and between nucleic acids and other nucleic acids [9]. Although nucleic acids are the main target of BPL, there is evidence demonstrating that BPL affects protein structures of influenza virus [10]. Also, it has been shown that BPL inactivated influenza viruses have a diminished ability to fuse with lipid membranes due to changes in the HA protein structure [11]. Nevertheless, formaldehyde inactivated influenza viruses have also been shown to lack the ability to fuse with lipid membranes [12]. Another method used to inactivate viruses is by the application of various forms of electromagnetic radiation. Inactivation is achieved through direct effects on nucleic acids, resulting in strand breaks, linkages, and nucleotide damage [13]. Initial reports on using gamma radiation to inactivate pathogens stated that a complete loss of influenza virus infectivity can be achieved after application of 0.65 kiloGrays (kGy) of gamma radiation, and this must be increased to 200 kGy before the HA protein structure is compromised [14]. This makes gamma radiation an effective way to inactivate influenza viruses without altering potential immunogenic proteins in the process. In the present study, we chose to compare three different methods for the inactivation of H9N2 AIV, namely formaldehyde, BPL, and gamma radiation. Immunogenicity of these preparations was assessed in a series of in vivo studies. In addition to the type of vaccine administered, vaccine adjuvants can also affect immune responses after vaccination. CpG ODN 2007 is a synthetic oligodeoxynucleotide recognized by TLR21 in chickens that has been shown to be an effective adjuvant when added to inactivated influenza virus chicken vaccines [15,16]. For this reason, CpG ODN 2007 was also combined with each of the H9N2 WIVs (formaldehyde, BPL and gamma radiation) to examine its effects on antibody- and cellmediated immune responses following vaccination in chickens with different H9N2 WIVs.
2. Material and methods 2.1. Chickens One-hundred and thirty, one-day-old specific pathogen free (SPF) chicks were purchased from the Canadian Food Inspection agency (Ottawa, Canada). The chickens were kept in the isolation facility at the Ontario Veterinary College, University of Guelph, Ontario. All sampling and treatment protocols were approved by the University of Guelph Animal Care Committee and were conducted with compliance to the guidelines provided by the Canadian Council on Animal Care. 2.2. Inactivation of H9N2 AIV H9N2 AIV (A/Turkey/Wisconsin/1/66) was propagated as previously described [17]. Inactivation with BPL and formaldehyde was performed as described previously [9,15]. Briefly, H9N2 virus was mixed with formaldehyde or BPL for 72 h at 37 °C (0.02%), or 30 min at 4 °C (0.1%), respectively. For gamma radiation of H9N2, concentrated H9N2 was lyophilized and subjected to 12.5 kGy of gamma radiation at the Southern Ontario Centre for Atmospheric Aerosol Research at the University of Toronto. Total protein concentration was determined for each WIV using a Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL) following the manufacturer’s recommendations. 2.3. CpG ODN 2007 CpG ODN 2007 was purchased from InvivoGen (San Diego, California, USA) and was resuspended in sterile phosphate buffered saline (PBS). 2.4. Vaccine formulation and experimental outline Chickens were divided into 10 groups summarized in Table 1. The abbreviated group names in Table 1 will be used to describe the vaccine which that group of chickens received. On day 7 post-hatch all chickens were vaccinated via intramuscular injection in the thigh muscle with 15 lg of one of the three WIVs. WIVs were administered alone, or in combination with an oil-emulsion adjuvant (AddaVaxTM) or 2 lg of CpG ODN 2007. On day 21 posthatch the chickens received a second vaccine dose. One group received PBS and served as a negative control group. Serum samples were collected weekly and spleens were harvested 10 days after the second vaccination. 2.5. Hemagglutination inhibition (HI) and enzyme-linked immunosorbent assay (ELISA) The HI assay was carried out as described previously [15]. For the ELISA, 96-well Maxisorp flat bottom plates were coated
Table 1 Vaccination groups. All birds were vaccinated on day 7 and 21 post-hatch. Group
Inactivated Virus
Adjuvant
1. PBS 2. Form 3. Form + Add 4. Form + CpG 5. BPL 6. BPL + Add 7. BPL + CpG 8. Gamma 9. Gamma + Add 10. Gamma + CpG
None 15 ug 15 ug 15 ug 15 ug 15 ug 15 ug 15 ug 15 ug 15 ug
None None AddaVaxTM 2 ug CpG ODN 2007 None AddaVaxTM 2 ug CpG ODN 2007 None AddaVaxTM 2 ug CpG ODN 2007
formaldehyde inactivated H9N2 formaldehyde inactivated H9N2 formaldehyde inactivated H9N2 beta-propiolactone inactivated (BPL) H9N2 BPL inactivated H9N2 BPL inactivated H9N2 gamma radiation inactivated H9N2 gamma radiation inactivated H9N2 gamma radiation inactivated H9N2
Please cite this article in press as: Astill J et al. Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens. Vaccine (2018), https://doi.org/10.1016/j. vaccine.2018.05.093
J. Astill et al. / Vaccine xxx (2018) xxx–xxx
overnight at 4 °C with 800 ng of heat-killed H9N2 virus in 100 ll per well. Plates were washed 3 times with wash buffer (0.05% Tween 20 in PBS) and blocked for 1 h at room temperature with blocking buffer (0.25% fish skin gelatin (Sigma-Aldrich) in wash buffer). Next, diluted serum samples were added to each well and incubated at room temperature for 1 h. Plates were washed 3 times and 100 ll of goat-anti chicken IgG or IgM conjugated to
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horseradish peroxidase (1/5000 in blocking buffer) was added to each well (Bethyl Laboratories, Montgomery, Texas). Plates were incubated at room temperature for 1 h, followed by 3 washes and then 100 ll of horseradish peroxidase substrate was added (ABTS peroxidase substrate system Kirkegaard and Perry Laboratories Gaithersburg, Maryland, USA). Colour development was stopped after 20 min with 100 ll of 1% SDS and absorbance at
Fig. 1. Serum HI antibody titers against H9N2 AIV. Average serum HI antibody titers from day 7, 14, 21, and 28 ppv. Secondary vaccination occurred on day 14 ppv immediately following blood collection. Chickens were vaccinated with 15 lg of one of the three H9N2 WIVs alone or with either AddaVaxTM or 2 lg of CpG ODN 2007. The negative control group that received PBS was removed from this figure as HI titers were zero at all time points. Standard error of the mean is indicated with error bars. Data were analyzed using one-way ANOVA followed by Duncan’s Multiple Range Test. Group means were compared at each time point, and differences in means were considered significant if p < 0.05. If no significant differences were noted, graphs appear without letter labels.
Fig. 2. Serum IgG antibody titers against H9N2 AIV. Average serum IgG antibody titers from (A) day 7, (B) day 14, (C) day 21, and (D) day 28 ppv. Secondary vaccination occurred on day 14 ppv immediately following blood collection. Chickens were vaccinated with 15 lg of one of the three H9N2 WIVs alone or with either AddaVaxTM or 2 lg of CpG ODN 2007. One group received just PBS as a negative control. Group means which share the same letter do not differ significantly. Standard error of the mean is indicated with error bars. Data were analyzed using one-way ANOVA followed by Duncan’s Multiple Range Test. Group means were compared at each time point, and differences in means were considered significant if p < 0.05. If no significant differences were noted, graphs appear without letter labels.
Please cite this article in press as: Astill J et al. Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens. Vaccine (2018), https://doi.org/10.1016/j. vaccine.2018.05.093
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405 nm was measured using a plate reader (Bio-Tek Instruments, Winooski, Vermont USA). Titers were estimated using a previously described method [18].
sion (72 °C for 10 s). Subsequent melt curve analysis was performed by heating to 95 °C for 10 s, cooling to 65 °C for 1 min, and heating to 97 °C. Primer sequences for b-actin, IFN-c, and IL2 can be found in [21,22], and [23], respectively.
2.6. Analysis of cell-mediated immune response 2.8. Statistical analysis Spleens were harvested from 5 chickens per group 10 days after the second vaccination and were suspended in ice cold HBSS immediately. Splenocyte single cell suspensions were prepared as described previously [19]. The cells were counted using a Moxi Z mini automated cell counter (Orflo Technologies) and seeded in 48-well flat bottom plates at a density of 5x106 cells/500 ll/well and incubated at 41 °C (5% CO2). Cells were then stimulated with the corresponding H9N2 WIV (1 lg/ml) that matched the vaccine groups. At 12 and 24 h post-stimulation the expression of IFN-c and Il-2 was measured. Supernatant was also collected 48 and 72 h post-stimulation and IFN-c was quantified using a chicken IFN-c sandwich ELISA kit following the manufacturer’s recommendations (InvitrogenTM). 2.7. Gene expression analysis RNA extraction and cDNA synthesis were performed as previously described [19]. RNA was extracted from splenocytes. Primers were synthesized by Sigma Aldrich. Relative expression of each gene was calculated relative to the chicken housekeeping gene bactin using software on the Light-CyclerÒ 480 II (Roche Diagnostics GmbH), as previously described [20]. Each reaction started with a pre-incubation at 95 °C for 10 min, followed by 45 cycles of denaturation (95 °C for 10 s), annealing (60 °C for 5 s) and exten-
Group means for antibody titers, IFN-c concentration, and relative gene expression were compared using Duncan’s Multiple Range Test using RÓ software and differences in means were considered significant if p < 0.05. 3. Results 3.1. Systemic antibody-mediated immune responses Serum was collected weekly following the primary vaccination until 28 days post-primary vaccination (ppv), totalling four time points to analyze systemic antibody-mediated immune responses. HI antibody titers were first analyzed from sera of vaccinated chickens. For non-adjuvanted WIVs, the Gamma vaccine induced significantly higher HI antibody titers on day 7 ppv (4-fold greater) than the BPL and Form vaccines (p < 0.05) (Fig. 1). The BPL vaccine induced significantly higher HI antibody titers compared to the Form vaccine on day 14 (4-fold greater) and 28 (2-fold greater) ppv (p < 0.05) (Fig. 1). Following day 7 ppv, the BPL and Gamma vaccines did not differ significantly in their respective induction of HI antibodies. When 2 mg of CpG ODN 2007 was included with any of the WIVs, it led to significant increases in HI antibody titers at various times ppv when compared to the WIVs administered
Fig. 3. Serum IgM antibody titers against H9N2 AIV. Average serum IgM antibody titers from (A) day 7, (B) day 14, (C) day 21, and (D) day 28 ppv. Secondary vaccination occurred on day 14 ppv immediately following blood collection. Chickens were vaccinated with 15 lg of one of the three H9N2 WIVs alone or with either AddaVaxTM or 2 lg of CpG ODN 2007. One group received just PBS as a negative control. Group means which share the same letter do not differ significantly. Standard error of the mean is indicated with error bars. Data were analyzed using one-way ANOVA followed by Duncan’s Multiple Range Test. Group means were compared at each time point, and differences in means were considered significant if p < 0.05. If no significant differences were noted, graphs appear without letter labels.
Please cite this article in press as: Astill J et al. Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens. Vaccine (2018), https://doi.org/10.1016/j. vaccine.2018.05.093
J. Astill et al. / Vaccine xxx (2018) xxx–xxx
alone or with AddaVaxTM (p < 0.05). Excluding day 14 ppv, all vaccines that contained CpG ODN 2007 (Form + CpG, BPL + CpG, and Gamma + CpG) induced the highest HI antibody titers relative to their counterpart WIV vaccines (no adjuvant or AddaVaxTM). Generally, (excluding day 14 ppv) there were no significant differences in HI antibody titer so long as 2 mg of CpG ODN 2007 was included in the vaccine formulation (p < 0.05) (Fig. 1). Interestingly, serum IgG antibody titers resembled HI antibody titers throughout the experiment. The Gamma vaccine induced significantly higher IgG antibody titers than the Form and Form + Add vaccines (23-fold and 6-fold greater, respectively) on day 7 ppv (p < 0.01), while the BPL vaccine induced higher IgG antibody titers than the Gamma vaccine on day 14, 21 and 28 ppv, and significantly higher IgG antibody titers than the Form vaccine on all time points ppv (p < 0.05) (Fig. 2). On day 21 and 28 ppv (day 7 and 14 post-secondary vaccination), serum IgG antibody titers were increased for all WIV vaccines when CpG ODN 2007 was included,
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relative to unadjuvanted or AddaVaxTM adjuvanted vaccines (Fig. 2c and d). This difference was significant (p < 0.05) for all groups except for the BPL WIV vaccine on day 21 ppv, as the BPL + CpG vaccine only induced significantly higher serum IgG antibody titers relative to the BPL vaccine (p < 0.05) (Fig. 2c). Finally, we checked serum IgM antibody titers following vaccination. The Gamma and BPL vaccines induced significantly higher IgM antibody titers (11-fold and 6-fold greater, respectively) relative to the Form vaccine on day 7 ppv (p < 0.01) (Fig. 3a), while the BPL vaccine induced significantly higher IgM antibody titers (5-fold and 3-fold greater, respectively) relative to the Form and Gamma vaccines on day 14 ppv (p < 0.05) (Fig. 3b). On day 21 ppv, all vaccines which contained CpG ODN 2007 induced significantly higher IgM antibody titers compared to nonadjuvanted or AddaVaxTM adjuvanted vaccines (p < 0.05), with one minor exception (Form + CpG and Form) (Fig. 3c). By day 28 ppv IgM antibody titers regressed, likely because of a switch to production of IgG isotype
Fig. 4. IFN-c concentration in supernatants of stimulated splenocytes. Splenocytes were stimulated with 1 lg/ml of BPL, formaldehyde, or gamma radiation inactivated H9N2 virus. Individual graphs represent groups of chickens vaccinated with either (A) formaldehyde inactivated H9N2, (C) BPL inactivated H9N2 or (E) gamma radiation inactivated H9N2, 48 h after stimulation. IFN-c in supernatant was also quantified 72 h post-stimulation for the same vaccine groups in (B), (D), and (F). Splenocytes from chickens which received PBS acted as controls and were stimulated and average IFN-c concentrations were compared to the vaccinated groups. Group means which share the same letter do not differ significantly. Standard error of the mean is indicated with error bars. Data were analyzed using one-way ANOVA followed by Duncan’s Multiple Range Test. Group means were compared at each time point, and differences in means were considered significant if p < 0.05. If no significant differences were noted, graphs appear without letter labels.
Please cite this article in press as: Astill J et al. Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens. Vaccine (2018), https://doi.org/10.1016/j. vaccine.2018.05.093
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antibodies, and only two groups (Form + CpG and Form) displayed titers significantly higher than non-vaccinated chickens (p < 0.05) (Fig. 3d). 3.2. Cell-mediated immune responses To analyze cell-mediated immune responses, chickens were euthanized 10 days post-secondary vaccination, and spleens were harvested. Splenocytes were re-stimulated in vitro with either formaldehyde, BPL, or gamma radiation inactivated H9N2 WIVs, using the same WIV that was used to vaccinate chickens. Interferon (IFN)-c production and IFN-c and interleukin (IL)-2 gene expression were quantified. IFN-c production in splenocytes from vaccinated chickens was elevated relative to non-vaccinated stimulated splenocytes (Fig. 4a–f). However, the only significant difference in IFN-c production occurred 72 h post-stimulation (p
< 0.05), as splenocytes from chickens vaccinated with the BPL + CpG vaccine produced significantly more IFN-c than splenocytes from chickens that were vaccinated with the PBS and BPL + Add vaccines (Fig. 4d). Relative expression of IFN-c reflected the initial observations on protein production (Fig. 5a–f). At both 12 and 24 h post-stimulation, a significant upregulation (minimum 7-fold increase and 4-fold increase, respectively) of IFN-c expression was noted in splenocytes from chickens which received the BPL + CpG vaccine relative to splenocytes from non-vaccinated chickens or chickens which received the BPL or BPL + Add vaccines (p < 0.05) (Fig. 5c and d). Similarly, IL-2 expression was upregulated in splenocytes from chickens which received the BPL + CpG vaccine. At 12 h post-stimulation, IL-2 expression in splenocytes from chickens which received the BPL + CpG vaccine was significantly higher than expression in splenocytes from chickens which received a control vaccine or received the BPL and BPL + Add
Fig. 5. IFN-c expression in stimulated splenocytes. Splenocytes were stimulated with 1 lg/ml of BPL, formaldehyde, or gamma radiation inactivated H9N2 virus. Individual graphs represent relative IFN-c expression 12 h post-stimulation from groups of chickens vaccinated with either (A) formaldehyde inactivated H9N2, (C) BPL inactivated H9N2, or (E) gamma radiation inactivated H9N2. IFN-c expression was also quantified after 24 h for the same vaccine groups in (B), (D), and (F). Splenocytes from the PBS group were stimulated and average IFN-c expression was compared to the vaccinated groups. Group means which share the same letter do not differ significantly. Standard error of the mean is indicated with error bars. Data were analyzed using one-way ANOVA followed by Duncan’s Multiple Range Test. Group means were compared at each time point, and differences in means were considered significant if p < 0.05. If no significant differences were noted, graphs appear without letter labels.
Please cite this article in press as: Astill J et al. Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens. Vaccine (2018), https://doi.org/10.1016/j. vaccine.2018.05.093
J. Astill et al. / Vaccine xxx (2018) xxx–xxx
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Fig. 6. IL-2 expression in stimulated splenocytes. Splenocytes were stimulated with 1 lg/ml of BPL, formaldehyde, or gamma radiation inactivated H9N2 virus. Individual graphs represent relative IL-2 expression 12 h post-stimulation from groups of chickens vaccinated with either (A) formaldehyde inactivated H9N2, (C) BPL inactivated H9N2, or (E) gamma radiation inactivated H9N2. IL-2 expression was also quantified after 24 h for the same vaccine groups in (B), (D), and (F). Splenocytes from the PBS group were stimulated and average IL-2 expression was compared to the vaccinated groups. Group means which share the same letter do not differ significantly. Standard error of the mean is indicated with error bars. Data were analyzed using one-way ANOVA followed by Duncan’s Multiple Range Test. Group means were compared at each time point, and differences in means were considered significant if p < 0.05. If no significant differences were noted, graphs appear without letter labels.
vaccines (p < 0.05) (Fig. 6c). We also observed a significant 3-fold upregulation of IL-2 expression in splenocytes from chickens which received the Form + CpG vaccine compared to splenocytes from chickens in the control group, at 24 h post-stimulation (p < 0.05) (Fig. 6b). We did not observe any other significant changes in IL-2 expression (Fig. 6a, d–f). 4. Discussion In the present study, we have formulated three different types of H9N2 WIVs and administered them to chickens intramuscularly in order to compare their immunogenicity. We chose to use formaldehyde, BPL, and gamma radiation to inactivate H9N2 AIV due to their unique mechanisms of virus inactivation. Antibody-mediated immune responses are crucial for defense against influenza virus infection. Antibodies curb influenza virus
replication by binding to viral proteins which in turn can inhibit their function [24]. We initially noted that the Gamma vaccine induced superior anti-H9N2 antibody titers compared to the Form and BPL vaccines. Following the first time point it became evident that the BPL vaccine was equal if not higher than the Gamma vaccine, and both were superior to the Form vaccine at inducing antibody-mediated immune responses. Formaldehyde can alter immunogenic proteins of influenza virus, in particular the HA protein, during inactivation [8]; this likely influenced antibodymediated immune responses after vaccination. In a study comparing influenza inactivation strategies, formaldehyde inactivation decreased the HA unit titer of an H9N2 virus by a factor of 4, while BPL treatment did not alter the HA unit titer at the same chemical concentration [9]. Both BPL and gamma radiation are thought to have minimal effect on protein structure, and in the current study, there were no consistent differences in antibody-mediated
Please cite this article in press as: Astill J et al. Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens. Vaccine (2018), https://doi.org/10.1016/j. vaccine.2018.05.093
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immune responses which favoured either inactivation method. However, BPL inactivation has practical and favourable characteristics compared to gamma radiation, as structural problems with gamma radiation inactivated influenza viruses have been reported after radiation of non-frozen virus preparations [25]. The present study also highlighted the capabilities of the TLR21 ligand, CpG ODN 2007, to act as an effective adjuvant when incorporated with all WIVs. It has been demonstrated that CpG ODN 2006 and 2007, which are both class B CpG ODNs, can enhance antibody-mediated immune responses when administered with inactivated influenza vaccines for chickens [15,16,26]. Cellular immune responses, mediated by CD4 + and CD8 + Tcells, are important components of the immunity against influenza [27]. CD8 + T-cells are major effector cells and kill influenza virus infected cells which display viral proteins on major histocompatibility complex (MHC)-I proteins, while CD4 + T-cells produce the necessary cytokines for the development of adaptive immune responses [28]. To assess the presence of cell-mediated immune responses, we stimulated splenocytes from vaccinated chickens with H9N2 WIVs and examined the recall response by analyzing the expression of IFN-c and IL-2 as well as the production of IFNc. We have shown here that the BPL + CpG vaccine induced a cell-mediated immune response in vaccinated chickens. However, we did not observe significant differences in IFN-c production for any other vaccine formulation. These results are in line with a previous study comparing BPL and formaldehyde WIV vaccines in mice which showed that a BPL WIV led to significantly higher levels of influenza nucleoprotein-specific CD8 + T-cells [29]. This difference was attributed to the ability of the BPL WIV to undergo endosomal membrane fusion in professional antigen presenting cells (APC). This allows influenza proteins to enter the cytoplasm and undergo cross-presentation, a process that lead to exogenous peptides being processed and presented via the MHC-I pathway, which is essential for the activation of CD8 + T-cells. CpG ODN 2007 could have also had a role in inducing cell-mediated immune responses. In chickens, TLR21 binding of CpG ODN 2007 in APCs leads to increased gene expression and production of proteins which are essential for T-cell activation, including co-stimulatory proteins such as CD80/86 [30]. In chickens the main cellular producers of IFN-c include CD8 + T-cells as well as CD4 + T helper 1 (Th1) cells [31]. Th1 cells produce certain cytokines including IFN-c and IL-2, which support cell-mediated immune responses [32]. We detected significantly increased IL-2 expression in those groups of chickens which received CpG ODN 2007 as vaccine adjuvant (Form + CpG and BPL + CpG). This suggests that CpG ODN 2007 could have led to the activation of Th1 cells. If CD8 + T-cells were also activated after vaccination, they could then become further activated by cytokine secreting Th1 cells. Given that BPL inactivated influenza WIVs promote CD8 + T-cell activation, it is understandable that cellmediated immune responses were induced by the BPL + CpG vaccine. However, IL-2 expression in the Form + CpG group was also elevated, but we did not observe any significant increases in IFNc production or expression. CpG ODN 2007 in the vaccine could have led to the activation of Th1 cells, but the formaldehyde WIV likely did not lead to the activation of CD8 + T-cells, perhaps because of the loss of WIV membrane fusion. In conclusion, we have demonstrated the effects that the inactivation method of an H9N2 virus can have on the immune response following vaccination in chickens. The addition of CpG ODN 2007 to the WIV vaccines increased serum antibody titers consistently, and when combined with the BPL WIV, evidence of cell-mediated immune responses were observed, indicating this formulation may be superior to the other vaccine formulations. Future studies should examine the effects that inactivation method has on the mucosal immune response following vaccination.
Acknowledgments We would like to thank the staff of the isolation unit at Ontario Veterinary College at the University of Guelph for their help with care and housing of chickens. This work was funded by Agriculture and Agri-Food Canada, the Ontario Ministry of Agriculture Food and Rural Affairs, Egg Farmers of Canada, Chicken Farmers of Saskatchewan, and the Canadian Poultry Research Council. This research is supported in part by the University of Guelph’s Food from Thought initiative, thanks to funding from the Canada First Research Excellence Fund. Author disclosure statement There is no conflict of interest regarding the publication of this paper. References [1] van Dijk JGB, Verhagen JH, Wille M, Waldenström J. Host and virus ecology as determinants of influenza A virus transmission in wild birds. Curr Opin Virol 2018;28:26–36. https://doi.org/10.1016/j.coviro.2017.10.006. [2] Gu M, Xu L, Wang X, Liu X. Current situation of H9N2 subtype avian influenza in China. Vet Res 2017;48:1–10. https://doi.org/10.1186/s13567-017-0453-2. [3] Shin JH, Mo JS, Kim JN, Mo IP, Ha B Do. Assessment of the safety and efficacy of low pathogenic avian influenza (H9N2) virus in inactivated oil emulsion vaccine in laying hens. J Vet Sci 2016;17:27–34. https://doi.org/10.4142/ jvs.2016.17.1.27. [4] Pawar SD, Tandale B V, Raut CG, Parkhi SS, Barde TD, Gurav YK, et al. Avian influenza H9N2 seroprevalence among poultry workers in Pune, India, 2010. PLoS One 2012;7. 10.1371/journal.pone.0036374. [5] Wang M, Fu C-X. Antibodies against H5 and H9 Avian Influenza among Poultry Workers in China. N Engl J Med 2009;360:2583–4. https://doi.org/10.1056/ NEJMc0900358. [6] Zhu H, Lam TT-Y, Smith DK, Guan Y. Emergence and development of H7N9 influenza viruses in China. Curr Opin Virol 2016;16:106–13.. [7] Krammer F, Palese P. Advances in the development of influenza virus vaccines. Nat Rev Drug Discov 2015;14:167–82. https://doi.org/10.1038/nrd4529. [8] Nunnally BK, Turula VE, Sitrin RD. Inactivated viral vaccines. Vaccine Anal Strateg Princ Control 2015:1–665. https://doi.org/10.1007/978-3-662-450246. [9] Pawar SD, Murtadak VB, Kale SD, Shinde PV, Parkhi SS. Evaluation of different inactivation methods for high and low pathogenic avian influenza viruses in egg-fluids for antigen preparation. J Virol Methods 2015;222:28–33. https:// doi.org/10.1016/j.jviromet.2015.05.004. [10] She YM, Cheng K, Farnsworth A, Li X, Cyr TD. Surface modifications of influenza proteins upon virus inactivation by b-propiolactone. Proteomics 2013;13:3537–47. https://doi.org/10.1002/pmic.201300096. [11] Desbat B, Lancelot E, Krell T, Nicolaï MC, Vogel F, Chevalier M, et al. Effect of the b-propiolactone treatment on the adsorption and fusion of influenza A/ Brisbane/59/2007 and A/New Caledonia/20/1999 virus H1N1 on a dimyristoylphosphatidylcholine/ganglioside GM3 mixed phospholipids monolayer at the air-water interface. Langmuir 2011;27:13675–83. https:// doi.org/10.1021/la2027175. [12] Geeraedts F, ter Veer W, Wilschut J, Huckriede A. Effect of viral membrane fusion activity on antibody induction by influenza H5N1 whole inactivated virus vaccine. Vaccine 2012;30:6501–7. https://doi.org/10.1016/ j.vaccine.2012.07.036. [13] Alsharifi M, Müllbacher A. The gamma-irradiated influenza vaccine and the prospect of producing safe vaccines in general. Immunol Cell Biol 2010;88:103–4. https://doi.org/10.1038/icb.2009.81. [14] International Atomic Energy Agency. Manual on radiation sterilization of medical and biological materials; 1973. [15] Singh SM, Alkie TN, Hodgins DC, Nagy É, Shojadoost B, Sharif S. Systemic immune responses to an inactivated, whole H9N2 avian influenza virus vaccine using class B CpG oligonucleotides in chickens. Vaccine 2015;33:3947–52. https://doi.org/10.1016/j.vaccine.2015.06.043. [16] St Paul M, Barjesteh N, Brisbin JT, Villaneueva AI, Read LR, Hodgins D, et al. Effects of ligands for Toll-like receptors 3, 4, and 21 as adjuvants on the immunogenicity of an avian influenza vaccine in chickens. Viral Immunol 2014;27:167–73. https://doi.org/10.1089/vim.2013.0124. [17] Yitbarek A, Alkie T, Taha-Abdelaziz K, Astill J, Rodriguez-Lecompte JC, Parkinson J, et al. Gut microbiota modulates type I interferon and antibodymediated immune responses in chickens infected with influenza virus subtype H9N2. Benef Microbes 2018;9:417–27. https://doi.org/10.3920/BM2017.0088. [18] Sacks JM, Gillette KG, Frank GH. Development and evaluation of an enzymelinked immunosorbent assay for bovine antibody to Pasteurella haemolytica: constructing an enzyme-linked immunosorbent assay titer. Am J Vet Res 1988;49:38–41.
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Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens Jake Astill a, Tamiru Alkie a,b, Alexander Yitbarek a, Khaled Taha Abd Abdelaziz c, Jegarubee Bavananthasivam a, Éva Nagy a, James John Petrik d, Shayan Sharif a,⇑ a
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada Department of Biology, Wilfred Laurier University, Waterloo, ON N2L 3C5, Canada1 c Pathology Department, Faculty of Veterinary Medicine, Beni-Suef University, Al Shamlah, 62511 Beni-Suef, Egypt d Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada b
a r t i c l e
i n f o
Article history: Received 2 March 2018 Received in revised form 20 May 2018 Accepted 21 May 2018 Available online xxxx Keywords: H9N2 AIV Whole inactivated virus vaccine Adaptive immunity Chickens Toll-like receptor 21 CpG ODN 2007
a b s t r a c t Several types of avian influenza virus (AIV) vaccines exist, including live-attenuated, vectored, and whole inactivated virus (WIV) vaccines. Inactivated vaccines offer some advantages compared to other types of vaccines, including ease of production and lack of ability to revert to a virulent state. However, WIV are poorly immunogenic, especially when these vaccines are delivered to mucosal surfaces. There are several factors that contribute to the immunogenicity of vaccines, one of which is the method used to inactivate viruses. Several methods exist for producing influenza WIVs, including formaldehyde, a chemical that affects protein structures leading to virus inactivation. Other methods include treatment with betapropiolactone (BPL) and the application of gamma radiation, both of which have less effects on protein structures compared to formaldehyde, and instead alter nucleic acids in the virion. Here, we sought to determine the effect of the above inactivation methods on immunogenicity of AIV vaccines. To this end, chickens were vaccinated with three different H9N2 WIVs using formaldehyde, BPL, and gamma radiation for inactivation. In addition to administering these three WIVs alone as vaccines, we also included CpG ODN 2007, a synthetic ligand recognized by Toll-like receptor (TLR)21 in chickens, as an adjuvant for each WIV. Subsequently, antibody- and cell-mediated immune responses were measured following vaccination. Antibody-mediated immune responses were increased in chickens that received the BPL and Gamma WIVs compared to the formaldehyde WIV. CpG ODN 2007 was found to significantly increase antibody responses for each WIV compared to WIV alone. Furthermore, we observed the presence of cell-mediated immune responses in chickens that received the BPL WIV combined with CpG ODN 2007. Based on these results, the BPL WIV + CpG ODN 2007 combination was the most effective vaccine at inducing adaptive immune responses against H9N2 AIV. Future studies should characterize mucosal adaptive immune responses to these vaccines. Ó 2018 Elsevier Ltd. All rights reserved.
1. Introduction Low pathogenic avian influenza viruses (LPAIV) are a class of avian influenza viruses (AIV) that commonly infect wild birds without causing any signs of clinical disease [1]. H9N2 AIV is a LPAIV that has become a concern for economical and health related reasons. H9N2 virus infection in chickens can decrease egg produc-
⇑ Corresponding author. 1
E-mail address: [email protected] (S. Sharif). Present address.
tion, and co-infection with other infectious pathogens can result in immunosuppression and pathological changes [2,3]. There is strong evidence of human exposure to the H9N2 virus among poultry workers in certain regions of the world including China and India [4,5], although not many cases of infection are reported. H9N2 AIV is also responsible for contributing its internal genes to a reassortant H7N9 virus which has demonstrated 40% mortality in human cases in China [6]. Vaccination is a potential strategy that can be used to control H9N2 infection in poultry. Many types of influenza vaccines exist, including live-attenuated vaccines, subunit vaccines, and
https://doi.org/10.1016/j.vaccine.2018.05.093 0264-410X/Ó 2018 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Astill J et al. Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens. Vaccine (2018), https://doi.org/10.1016/j. vaccine.2018.05.093
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inactivated vaccines. Whole-inactivated virus (WIV) vaccines for influenza were first explored in the 1940s, when formaldehyde and other compounds were used to inactivate influenza viruses [7]. Since then, many different ways to produce influenza WIV vaccines have emerged, including other chemical treatments or the application of different forms of electromagnetic radiation [8]. Two common chemicals used to produce influenza WIVs include formaldehyde and beta-propiolactone (BPL). As an aldehyde, formaldehyde reacts with protein structures, crosslinking various amino acids. This in turn affects the function of proteins and can lead to a loss of virus infectivity [9]. Altering protein structures is an effective way to induce virus inactivation, however, this can change the outcome of the elicited immune response following vaccination due to changes on epitopes of immunogenic proteins, such as the influenza hemagglutinin (HA) protein [9]. BPL is an electrophilic compound that reacts with nucleic acids, mainly with the nucleotides guanosine and adenosine [8]. These reactions alter the structure of nucleic acids inducing changes such as strand breaks and improper linkages between nucleic acids and protein, and between nucleic acids and other nucleic acids [9]. Although nucleic acids are the main target of BPL, there is evidence demonstrating that BPL affects protein structures of influenza virus [10]. Also, it has been shown that BPL inactivated influenza viruses have a diminished ability to fuse with lipid membranes due to changes in the HA protein structure [11]. Nevertheless, formaldehyde inactivated influenza viruses have also been shown to lack the ability to fuse with lipid membranes [12]. Another method used to inactivate viruses is by the application of various forms of electromagnetic radiation. Inactivation is achieved through direct effects on nucleic acids, resulting in strand breaks, linkages, and nucleotide damage [13]. Initial reports on using gamma radiation to inactivate pathogens stated that a complete loss of influenza virus infectivity can be achieved after application of 0.65 kiloGrays (kGy) of gamma radiation, and this must be increased to 200 kGy before the HA protein structure is compromised [14]. This makes gamma radiation an effective way to inactivate influenza viruses without altering potential immunogenic proteins in the process. In the present study, we chose to compare three different methods for the inactivation of H9N2 AIV, namely formaldehyde, BPL, and gamma radiation. Immunogenicity of these preparations was assessed in a series of in vivo studies. In addition to the type of vaccine administered, vaccine adjuvants can also affect immune responses after vaccination. CpG ODN 2007 is a synthetic oligodeoxynucleotide recognized by TLR21 in chickens that has been shown to be an effective adjuvant when added to inactivated influenza virus chicken vaccines [15,16]. For this reason, CpG ODN 2007 was also combined with each of the H9N2 WIVs (formaldehyde, BPL and gamma radiation) to examine its effects on antibody- and cellmediated immune responses following vaccination in chickens with different H9N2 WIVs.
2. Material and methods 2.1. Chickens One-hundred and thirty, one-day-old specific pathogen free (SPF) chicks were purchased from the Canadian Food Inspection agency (Ottawa, Canada). The chickens were kept in the isolation facility at the Ontario Veterinary College, University of Guelph, Ontario. All sampling and treatment protocols were approved by the University of Guelph Animal Care Committee and were conducted with compliance to the guidelines provided by the Canadian Council on Animal Care. 2.2. Inactivation of H9N2 AIV H9N2 AIV (A/Turkey/Wisconsin/1/66) was propagated as previously described [17]. Inactivation with BPL and formaldehyde was performed as described previously [9,15]. Briefly, H9N2 virus was mixed with formaldehyde or BPL for 72 h at 37 °C (0.02%), or 30 min at 4 °C (0.1%), respectively. For gamma radiation of H9N2, concentrated H9N2 was lyophilized and subjected to 12.5 kGy of gamma radiation at the Southern Ontario Centre for Atmospheric Aerosol Research at the University of Toronto. Total protein concentration was determined for each WIV using a Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL) following the manufacturer’s recommendations. 2.3. CpG ODN 2007 CpG ODN 2007 was purchased from InvivoGen (San Diego, California, USA) and was resuspended in sterile phosphate buffered saline (PBS). 2.4. Vaccine formulation and experimental outline Chickens were divided into 10 groups summarized in Table 1. The abbreviated group names in Table 1 will be used to describe the vaccine which that group of chickens received. On day 7 post-hatch all chickens were vaccinated via intramuscular injection in the thigh muscle with 15 lg of one of the three WIVs. WIVs were administered alone, or in combination with an oil-emulsion adjuvant (AddaVaxTM) or 2 lg of CpG ODN 2007. On day 21 posthatch the chickens received a second vaccine dose. One group received PBS and served as a negative control group. Serum samples were collected weekly and spleens were harvested 10 days after the second vaccination. 2.5. Hemagglutination inhibition (HI) and enzyme-linked immunosorbent assay (ELISA) The HI assay was carried out as described previously [15]. For the ELISA, 96-well Maxisorp flat bottom plates were coated
Table 1 Vaccination groups. All birds were vaccinated on day 7 and 21 post-hatch. Group
Inactivated Virus
Adjuvant
1. PBS 2. Form 3. Form + Add 4. Form + CpG 5. BPL 6. BPL + Add 7. BPL + CpG 8. Gamma 9. Gamma + Add 10. Gamma + CpG
None 15 ug 15 ug 15 ug 15 ug 15 ug 15 ug 15 ug 15 ug 15 ug
None None AddaVaxTM 2 ug CpG ODN 2007 None AddaVaxTM 2 ug CpG ODN 2007 None AddaVaxTM 2 ug CpG ODN 2007
formaldehyde inactivated H9N2 formaldehyde inactivated H9N2 formaldehyde inactivated H9N2 beta-propiolactone inactivated (BPL) H9N2 BPL inactivated H9N2 BPL inactivated H9N2 gamma radiation inactivated H9N2 gamma radiation inactivated H9N2 gamma radiation inactivated H9N2
Please cite this article in press as: Astill J et al. Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens. Vaccine (2018), https://doi.org/10.1016/j. vaccine.2018.05.093
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overnight at 4 °C with 800 ng of heat-killed H9N2 virus in 100 ll per well. Plates were washed 3 times with wash buffer (0.05% Tween 20 in PBS) and blocked for 1 h at room temperature with blocking buffer (0.25% fish skin gelatin (Sigma-Aldrich) in wash buffer). Next, diluted serum samples were added to each well and incubated at room temperature for 1 h. Plates were washed 3 times and 100 ll of goat-anti chicken IgG or IgM conjugated to
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horseradish peroxidase (1/5000 in blocking buffer) was added to each well (Bethyl Laboratories, Montgomery, Texas). Plates were incubated at room temperature for 1 h, followed by 3 washes and then 100 ll of horseradish peroxidase substrate was added (ABTS peroxidase substrate system Kirkegaard and Perry Laboratories Gaithersburg, Maryland, USA). Colour development was stopped after 20 min with 100 ll of 1% SDS and absorbance at
Fig. 1. Serum HI antibody titers against H9N2 AIV. Average serum HI antibody titers from day 7, 14, 21, and 28 ppv. Secondary vaccination occurred on day 14 ppv immediately following blood collection. Chickens were vaccinated with 15 lg of one of the three H9N2 WIVs alone or with either AddaVaxTM or 2 lg of CpG ODN 2007. The negative control group that received PBS was removed from this figure as HI titers were zero at all time points. Standard error of the mean is indicated with error bars. Data were analyzed using one-way ANOVA followed by Duncan’s Multiple Range Test. Group means were compared at each time point, and differences in means were considered significant if p < 0.05. If no significant differences were noted, graphs appear without letter labels.
Fig. 2. Serum IgG antibody titers against H9N2 AIV. Average serum IgG antibody titers from (A) day 7, (B) day 14, (C) day 21, and (D) day 28 ppv. Secondary vaccination occurred on day 14 ppv immediately following blood collection. Chickens were vaccinated with 15 lg of one of the three H9N2 WIVs alone or with either AddaVaxTM or 2 lg of CpG ODN 2007. One group received just PBS as a negative control. Group means which share the same letter do not differ significantly. Standard error of the mean is indicated with error bars. Data were analyzed using one-way ANOVA followed by Duncan’s Multiple Range Test. Group means were compared at each time point, and differences in means were considered significant if p < 0.05. If no significant differences were noted, graphs appear without letter labels.
Please cite this article in press as: Astill J et al. Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens. Vaccine (2018), https://doi.org/10.1016/j. vaccine.2018.05.093
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405 nm was measured using a plate reader (Bio-Tek Instruments, Winooski, Vermont USA). Titers were estimated using a previously described method [18].
sion (72 °C for 10 s). Subsequent melt curve analysis was performed by heating to 95 °C for 10 s, cooling to 65 °C for 1 min, and heating to 97 °C. Primer sequences for b-actin, IFN-c, and IL2 can be found in [21,22], and [23], respectively.
2.6. Analysis of cell-mediated immune response 2.8. Statistical analysis Spleens were harvested from 5 chickens per group 10 days after the second vaccination and were suspended in ice cold HBSS immediately. Splenocyte single cell suspensions were prepared as described previously [19]. The cells were counted using a Moxi Z mini automated cell counter (Orflo Technologies) and seeded in 48-well flat bottom plates at a density of 5x106 cells/500 ll/well and incubated at 41 °C (5% CO2). Cells were then stimulated with the corresponding H9N2 WIV (1 lg/ml) that matched the vaccine groups. At 12 and 24 h post-stimulation the expression of IFN-c and Il-2 was measured. Supernatant was also collected 48 and 72 h post-stimulation and IFN-c was quantified using a chicken IFN-c sandwich ELISA kit following the manufacturer’s recommendations (InvitrogenTM). 2.7. Gene expression analysis RNA extraction and cDNA synthesis were performed as previously described [19]. RNA was extracted from splenocytes. Primers were synthesized by Sigma Aldrich. Relative expression of each gene was calculated relative to the chicken housekeeping gene bactin using software on the Light-CyclerÒ 480 II (Roche Diagnostics GmbH), as previously described [20]. Each reaction started with a pre-incubation at 95 °C for 10 min, followed by 45 cycles of denaturation (95 °C for 10 s), annealing (60 °C for 5 s) and exten-
Group means for antibody titers, IFN-c concentration, and relative gene expression were compared using Duncan’s Multiple Range Test using RÓ software and differences in means were considered significant if p < 0.05. 3. Results 3.1. Systemic antibody-mediated immune responses Serum was collected weekly following the primary vaccination until 28 days post-primary vaccination (ppv), totalling four time points to analyze systemic antibody-mediated immune responses. HI antibody titers were first analyzed from sera of vaccinated chickens. For non-adjuvanted WIVs, the Gamma vaccine induced significantly higher HI antibody titers on day 7 ppv (4-fold greater) than the BPL and Form vaccines (p < 0.05) (Fig. 1). The BPL vaccine induced significantly higher HI antibody titers compared to the Form vaccine on day 14 (4-fold greater) and 28 (2-fold greater) ppv (p < 0.05) (Fig. 1). Following day 7 ppv, the BPL and Gamma vaccines did not differ significantly in their respective induction of HI antibodies. When 2 mg of CpG ODN 2007 was included with any of the WIVs, it led to significant increases in HI antibody titers at various times ppv when compared to the WIVs administered
Fig. 3. Serum IgM antibody titers against H9N2 AIV. Average serum IgM antibody titers from (A) day 7, (B) day 14, (C) day 21, and (D) day 28 ppv. Secondary vaccination occurred on day 14 ppv immediately following blood collection. Chickens were vaccinated with 15 lg of one of the three H9N2 WIVs alone or with either AddaVaxTM or 2 lg of CpG ODN 2007. One group received just PBS as a negative control. Group means which share the same letter do not differ significantly. Standard error of the mean is indicated with error bars. Data were analyzed using one-way ANOVA followed by Duncan’s Multiple Range Test. Group means were compared at each time point, and differences in means were considered significant if p < 0.05. If no significant differences were noted, graphs appear without letter labels.
Please cite this article in press as: Astill J et al. Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens. Vaccine (2018), https://doi.org/10.1016/j. vaccine.2018.05.093
J. Astill et al. / Vaccine xxx (2018) xxx–xxx
alone or with AddaVaxTM (p < 0.05). Excluding day 14 ppv, all vaccines that contained CpG ODN 2007 (Form + CpG, BPL + CpG, and Gamma + CpG) induced the highest HI antibody titers relative to their counterpart WIV vaccines (no adjuvant or AddaVaxTM). Generally, (excluding day 14 ppv) there were no significant differences in HI antibody titer so long as 2 mg of CpG ODN 2007 was included in the vaccine formulation (p < 0.05) (Fig. 1). Interestingly, serum IgG antibody titers resembled HI antibody titers throughout the experiment. The Gamma vaccine induced significantly higher IgG antibody titers than the Form and Form + Add vaccines (23-fold and 6-fold greater, respectively) on day 7 ppv (p < 0.01), while the BPL vaccine induced higher IgG antibody titers than the Gamma vaccine on day 14, 21 and 28 ppv, and significantly higher IgG antibody titers than the Form vaccine on all time points ppv (p < 0.05) (Fig. 2). On day 21 and 28 ppv (day 7 and 14 post-secondary vaccination), serum IgG antibody titers were increased for all WIV vaccines when CpG ODN 2007 was included,
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relative to unadjuvanted or AddaVaxTM adjuvanted vaccines (Fig. 2c and d). This difference was significant (p < 0.05) for all groups except for the BPL WIV vaccine on day 21 ppv, as the BPL + CpG vaccine only induced significantly higher serum IgG antibody titers relative to the BPL vaccine (p < 0.05) (Fig. 2c). Finally, we checked serum IgM antibody titers following vaccination. The Gamma and BPL vaccines induced significantly higher IgM antibody titers (11-fold and 6-fold greater, respectively) relative to the Form vaccine on day 7 ppv (p < 0.01) (Fig. 3a), while the BPL vaccine induced significantly higher IgM antibody titers (5-fold and 3-fold greater, respectively) relative to the Form and Gamma vaccines on day 14 ppv (p < 0.05) (Fig. 3b). On day 21 ppv, all vaccines which contained CpG ODN 2007 induced significantly higher IgM antibody titers compared to nonadjuvanted or AddaVaxTM adjuvanted vaccines (p < 0.05), with one minor exception (Form + CpG and Form) (Fig. 3c). By day 28 ppv IgM antibody titers regressed, likely because of a switch to production of IgG isotype
Fig. 4. IFN-c concentration in supernatants of stimulated splenocytes. Splenocytes were stimulated with 1 lg/ml of BPL, formaldehyde, or gamma radiation inactivated H9N2 virus. Individual graphs represent groups of chickens vaccinated with either (A) formaldehyde inactivated H9N2, (C) BPL inactivated H9N2 or (E) gamma radiation inactivated H9N2, 48 h after stimulation. IFN-c in supernatant was also quantified 72 h post-stimulation for the same vaccine groups in (B), (D), and (F). Splenocytes from chickens which received PBS acted as controls and were stimulated and average IFN-c concentrations were compared to the vaccinated groups. Group means which share the same letter do not differ significantly. Standard error of the mean is indicated with error bars. Data were analyzed using one-way ANOVA followed by Duncan’s Multiple Range Test. Group means were compared at each time point, and differences in means were considered significant if p < 0.05. If no significant differences were noted, graphs appear without letter labels.
Please cite this article in press as: Astill J et al. Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens. Vaccine (2018), https://doi.org/10.1016/j. vaccine.2018.05.093
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antibodies, and only two groups (Form + CpG and Form) displayed titers significantly higher than non-vaccinated chickens (p < 0.05) (Fig. 3d). 3.2. Cell-mediated immune responses To analyze cell-mediated immune responses, chickens were euthanized 10 days post-secondary vaccination, and spleens were harvested. Splenocytes were re-stimulated in vitro with either formaldehyde, BPL, or gamma radiation inactivated H9N2 WIVs, using the same WIV that was used to vaccinate chickens. Interferon (IFN)-c production and IFN-c and interleukin (IL)-2 gene expression were quantified. IFN-c production in splenocytes from vaccinated chickens was elevated relative to non-vaccinated stimulated splenocytes (Fig. 4a–f). However, the only significant difference in IFN-c production occurred 72 h post-stimulation (p
< 0.05), as splenocytes from chickens vaccinated with the BPL + CpG vaccine produced significantly more IFN-c than splenocytes from chickens that were vaccinated with the PBS and BPL + Add vaccines (Fig. 4d). Relative expression of IFN-c reflected the initial observations on protein production (Fig. 5a–f). At both 12 and 24 h post-stimulation, a significant upregulation (minimum 7-fold increase and 4-fold increase, respectively) of IFN-c expression was noted in splenocytes from chickens which received the BPL + CpG vaccine relative to splenocytes from non-vaccinated chickens or chickens which received the BPL or BPL + Add vaccines (p < 0.05) (Fig. 5c and d). Similarly, IL-2 expression was upregulated in splenocytes from chickens which received the BPL + CpG vaccine. At 12 h post-stimulation, IL-2 expression in splenocytes from chickens which received the BPL + CpG vaccine was significantly higher than expression in splenocytes from chickens which received a control vaccine or received the BPL and BPL + Add
Fig. 5. IFN-c expression in stimulated splenocytes. Splenocytes were stimulated with 1 lg/ml of BPL, formaldehyde, or gamma radiation inactivated H9N2 virus. Individual graphs represent relative IFN-c expression 12 h post-stimulation from groups of chickens vaccinated with either (A) formaldehyde inactivated H9N2, (C) BPL inactivated H9N2, or (E) gamma radiation inactivated H9N2. IFN-c expression was also quantified after 24 h for the same vaccine groups in (B), (D), and (F). Splenocytes from the PBS group were stimulated and average IFN-c expression was compared to the vaccinated groups. Group means which share the same letter do not differ significantly. Standard error of the mean is indicated with error bars. Data were analyzed using one-way ANOVA followed by Duncan’s Multiple Range Test. Group means were compared at each time point, and differences in means were considered significant if p < 0.05. If no significant differences were noted, graphs appear without letter labels.
Please cite this article in press as: Astill J et al. Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens. Vaccine (2018), https://doi.org/10.1016/j. vaccine.2018.05.093
J. Astill et al. / Vaccine xxx (2018) xxx–xxx
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Fig. 6. IL-2 expression in stimulated splenocytes. Splenocytes were stimulated with 1 lg/ml of BPL, formaldehyde, or gamma radiation inactivated H9N2 virus. Individual graphs represent relative IL-2 expression 12 h post-stimulation from groups of chickens vaccinated with either (A) formaldehyde inactivated H9N2, (C) BPL inactivated H9N2, or (E) gamma radiation inactivated H9N2. IL-2 expression was also quantified after 24 h for the same vaccine groups in (B), (D), and (F). Splenocytes from the PBS group were stimulated and average IL-2 expression was compared to the vaccinated groups. Group means which share the same letter do not differ significantly. Standard error of the mean is indicated with error bars. Data were analyzed using one-way ANOVA followed by Duncan’s Multiple Range Test. Group means were compared at each time point, and differences in means were considered significant if p < 0.05. If no significant differences were noted, graphs appear without letter labels.
vaccines (p < 0.05) (Fig. 6c). We also observed a significant 3-fold upregulation of IL-2 expression in splenocytes from chickens which received the Form + CpG vaccine compared to splenocytes from chickens in the control group, at 24 h post-stimulation (p < 0.05) (Fig. 6b). We did not observe any other significant changes in IL-2 expression (Fig. 6a, d–f). 4. Discussion In the present study, we have formulated three different types of H9N2 WIVs and administered them to chickens intramuscularly in order to compare their immunogenicity. We chose to use formaldehyde, BPL, and gamma radiation to inactivate H9N2 AIV due to their unique mechanisms of virus inactivation. Antibody-mediated immune responses are crucial for defense against influenza virus infection. Antibodies curb influenza virus
replication by binding to viral proteins which in turn can inhibit their function [24]. We initially noted that the Gamma vaccine induced superior anti-H9N2 antibody titers compared to the Form and BPL vaccines. Following the first time point it became evident that the BPL vaccine was equal if not higher than the Gamma vaccine, and both were superior to the Form vaccine at inducing antibody-mediated immune responses. Formaldehyde can alter immunogenic proteins of influenza virus, in particular the HA protein, during inactivation [8]; this likely influenced antibodymediated immune responses after vaccination. In a study comparing influenza inactivation strategies, formaldehyde inactivation decreased the HA unit titer of an H9N2 virus by a factor of 4, while BPL treatment did not alter the HA unit titer at the same chemical concentration [9]. Both BPL and gamma radiation are thought to have minimal effect on protein structure, and in the current study, there were no consistent differences in antibody-mediated
Please cite this article in press as: Astill J et al. Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens. Vaccine (2018), https://doi.org/10.1016/j. vaccine.2018.05.093
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immune responses which favoured either inactivation method. However, BPL inactivation has practical and favourable characteristics compared to gamma radiation, as structural problems with gamma radiation inactivated influenza viruses have been reported after radiation of non-frozen virus preparations [25]. The present study also highlighted the capabilities of the TLR21 ligand, CpG ODN 2007, to act as an effective adjuvant when incorporated with all WIVs. It has been demonstrated that CpG ODN 2006 and 2007, which are both class B CpG ODNs, can enhance antibody-mediated immune responses when administered with inactivated influenza vaccines for chickens [15,16,26]. Cellular immune responses, mediated by CD4 + and CD8 + Tcells, are important components of the immunity against influenza [27]. CD8 + T-cells are major effector cells and kill influenza virus infected cells which display viral proteins on major histocompatibility complex (MHC)-I proteins, while CD4 + T-cells produce the necessary cytokines for the development of adaptive immune responses [28]. To assess the presence of cell-mediated immune responses, we stimulated splenocytes from vaccinated chickens with H9N2 WIVs and examined the recall response by analyzing the expression of IFN-c and IL-2 as well as the production of IFNc. We have shown here that the BPL + CpG vaccine induced a cell-mediated immune response in vaccinated chickens. However, we did not observe significant differences in IFN-c production for any other vaccine formulation. These results are in line with a previous study comparing BPL and formaldehyde WIV vaccines in mice which showed that a BPL WIV led to significantly higher levels of influenza nucleoprotein-specific CD8 + T-cells [29]. This difference was attributed to the ability of the BPL WIV to undergo endosomal membrane fusion in professional antigen presenting cells (APC). This allows influenza proteins to enter the cytoplasm and undergo cross-presentation, a process that lead to exogenous peptides being processed and presented via the MHC-I pathway, which is essential for the activation of CD8 + T-cells. CpG ODN 2007 could have also had a role in inducing cell-mediated immune responses. In chickens, TLR21 binding of CpG ODN 2007 in APCs leads to increased gene expression and production of proteins which are essential for T-cell activation, including co-stimulatory proteins such as CD80/86 [30]. In chickens the main cellular producers of IFN-c include CD8 + T-cells as well as CD4 + T helper 1 (Th1) cells [31]. Th1 cells produce certain cytokines including IFN-c and IL-2, which support cell-mediated immune responses [32]. We detected significantly increased IL-2 expression in those groups of chickens which received CpG ODN 2007 as vaccine adjuvant (Form + CpG and BPL + CpG). This suggests that CpG ODN 2007 could have led to the activation of Th1 cells. If CD8 + T-cells were also activated after vaccination, they could then become further activated by cytokine secreting Th1 cells. Given that BPL inactivated influenza WIVs promote CD8 + T-cell activation, it is understandable that cellmediated immune responses were induced by the BPL + CpG vaccine. However, IL-2 expression in the Form + CpG group was also elevated, but we did not observe any significant increases in IFNc production or expression. CpG ODN 2007 in the vaccine could have led to the activation of Th1 cells, but the formaldehyde WIV likely did not lead to the activation of CD8 + T-cells, perhaps because of the loss of WIV membrane fusion. In conclusion, we have demonstrated the effects that the inactivation method of an H9N2 virus can have on the immune response following vaccination in chickens. The addition of CpG ODN 2007 to the WIV vaccines increased serum antibody titers consistently, and when combined with the BPL WIV, evidence of cell-mediated immune responses were observed, indicating this formulation may be superior to the other vaccine formulations. Future studies should examine the effects that inactivation method has on the mucosal immune response following vaccination.
Acknowledgments We would like to thank the staff of the isolation unit at Ontario Veterinary College at the University of Guelph for their help with care and housing of chickens. This work was funded by Agriculture and Agri-Food Canada, the Ontario Ministry of Agriculture Food and Rural Affairs, Egg Farmers of Canada, Chicken Farmers of Saskatchewan, and the Canadian Poultry Research Council. This research is supported in part by the University of Guelph’s Food from Thought initiative, thanks to funding from the Canada First Research Excellence Fund. Author disclosure statement There is no conflict of interest regarding the publication of this paper. References [1] van Dijk JGB, Verhagen JH, Wille M, Waldenström J. Host and virus ecology as determinants of influenza A virus transmission in wild birds. Curr Opin Virol 2018;28:26–36. https://doi.org/10.1016/j.coviro.2017.10.006. [2] Gu M, Xu L, Wang X, Liu X. Current situation of H9N2 subtype avian influenza in China. Vet Res 2017;48:1–10. https://doi.org/10.1186/s13567-017-0453-2. [3] Shin JH, Mo JS, Kim JN, Mo IP, Ha B Do. Assessment of the safety and efficacy of low pathogenic avian influenza (H9N2) virus in inactivated oil emulsion vaccine in laying hens. J Vet Sci 2016;17:27–34. https://doi.org/10.4142/ jvs.2016.17.1.27. [4] Pawar SD, Tandale B V, Raut CG, Parkhi SS, Barde TD, Gurav YK, et al. Avian influenza H9N2 seroprevalence among poultry workers in Pune, India, 2010. PLoS One 2012;7. 10.1371/journal.pone.0036374. [5] Wang M, Fu C-X. Antibodies against H5 and H9 Avian Influenza among Poultry Workers in China. N Engl J Med 2009;360:2583–4. https://doi.org/10.1056/ NEJMc0900358. [6] Zhu H, Lam TT-Y, Smith DK, Guan Y. Emergence and development of H7N9 influenza viruses in China. Curr Opin Virol 2016;16:106–13.
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Please cite this article in press as: Astill J et al. Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens. Vaccine (2018), https://doi.org/10.1016/j. vaccine.2018.05.093