Humoral response and antiviral cytokine expression following vaccination of thoroughbred weanlings—A blinded comparison of commercially available vaccines

Humoral response and antiviral cytokine expression following vaccination of thoroughbred weanlings—A blinded comparison of commercially available vaccines

Vaccine 31 (2013) 5216–5222 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine Humoral response an...

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Vaccine 31 (2013) 5216–5222

Contents lists available at ScienceDirect

Vaccine journal homepage: www.elsevier.com/locate/vaccine

Humoral response and antiviral cytokine expression following vaccination of thoroughbred weanlings—A blinded comparison of commercially available vaccines Sarah Gildea a , Michelle Quinlivan a , Barbara A. Murphy b , Ann Cullinane a,∗ a b

Virology Unit, The Irish Equine Centre, Johnstown, Naas, Co., Kildare, Ireland School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland

a r t i c l e

i n f o

Article history: Received 13 May 2013 Received in revised form 19 August 2013 Accepted 27 August 2013 Available online 8 September 2013 Keywords: Equine influenza Vaccine Comparison Antibody Cell mediated immunity Thoroughbred

a b s t r a c t Previous studies in experimental ponies using interferon gamma (IFN-) as a marker for cell mediated immune (CMI) response demonstrated an increase in IFN- gene expression following vaccination with an ISCOM subunit, a canarypox recombinant and more recently, an inactivated whole virus vaccine. The objective of this study was to carry out an independent comparison of both humoral antibody and CMI responses elicited following vaccination with all these vaccine presentation systems. Antibody response of 44 Thoroughbred weanlings was monitored for three weeks following the second dose of primary vaccination (V2) by single radial haemolysis (SRH). The pattern of antibody response was similar for all vaccines. The antibody response of horses vaccinated with the inactivated whole virus vaccine (Duvaxyn IE-T Plus) was superior to that of the horses vaccinated with the ISCOM-matrix subunit (Equilis Prequenza Te) and canarypox recombinant (ProteqFlu-Te) vaccine. In this study 39% of weanlings failed to seroconvert following their first dose of primary vaccination (V1). Poor response to vaccination (H3N8) was observed among weanlings vaccinated with Equilis Prequenza Te and ProteqFlu-Te but not among those vaccinated with Duvaxyn IE-T Plus. PAXgene bloods were collected on days 0, 2, 7 and 14 following V1. Gene expression levels of IFN-, IL-1␤ (proinflammatory cytokine) and IL-4 (B cell stimulating cytokine) were measured using RT-PCR. Mean gene expression levels of IL-1␤ and IL-4 peaked on day 14 post vaccination. The increase in IL-4 gene expression by horses vaccinated with Equilis Prequenza Te was significantly greater to those vaccinated with the other two products. Vaccination with all three vaccines resulted in a significant increase in IFN- gene expression which peaked at 7 days post V1. Overall, there was no significant difference in IFN- gene expression by the horses vaccinated with the whole inactivated, the subunit and the canarypox recombinant vaccines included in this study. © 2013 Published by Elsevier Ltd.

1. Introduction Equine influenza (EI) is endemic in Europe and North America and outbreaks of major economic significance have been reported on all continents. Despite the many different combinations of haemagglutinin (HA) and neuraminidase subtypes that occur in birds, only subtypes H7N7 and H3N8 have ever established in horses. H7N7 viruses caused outbreaks of influenza in horses for over two decades but are now considered extinct [1]. Since 1979 all outbreaks of EI for which there are virus isolates, have been attributed to H3N8 and the OIE (Office International des Epizooties) stipulates that there is no requirement for inclusion of a H7N7 virus in vaccines [2]. However, the majority of commercially available vaccines contain both virus subtypes. Vaccination against EI is an

∗ Corresponding author. Tel.: +353 45 866266; fax: +353 45 866273. E-mail address: [email protected] (A. Cullinane). 0264-410X/$ – see front matter © 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.vaccine.2013.08.083

effective method of disease control in many countries worldwide. In countries where equine influenza virus (EIV) is endemic, vaccination minimises the incidence of disease and dissemination of virus at equestrian events. Since the introduction of mandatory EI vaccination of race horses in Ireland and the UK in the early 1980s, no race meeting or major equestrian event has been cancelled in either country as a result of the disease [3]. In countries where the virus is not endemic, vaccination in conjunction with quarantine are the barriers which prevent an incursion of the virus. Notwithstanding, in the past twenty five years major outbreaks have occurred in South Africa (1986 and 2003), India (1987), Hong Kong (1992) and more recently Australia (2007) following the international movement of horses vaccinated with suboptimal products that appear to have induced clinical but not virological protection [4–8]. Vaccine evaluations are carried out by vaccine companies or at the request of vaccine companies for regulatory or marketing purposes. These studies are usually performed under optimal conditions for vaccination and predominately in experimental ponies

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rather than target animals in the field. In order to best evaluate humoral response following vaccination, serum antibodies generated against the virus HA are commonly measured using the single radial haemolysis (SRH) test. These antibodies neutralise virus infectivity and antibody levels correlating with protection have been established in both experimental challenge studies and in the field [9–12]. A recent independent comparative vaccine study carried out in seronegative Thoroughbred weanlings demonstrated a significant difference in SRH antibody levels following vaccination with commercially available products [13]. Such independent evaluations are carried out infrequently and are essential for equivalent comparison between products. While it is widely accepted that humoral antibody levels are fundamental for protection, there is evidence that immune mechanisms other than serum antibody may play a role in the prevention of disease. Protection against experimental challenge with EI was demonstrated six months after a single intranasal administration of a cold-adapted, modified live EI vaccine despite the absence of high antibody levels [14]. Other studies have shown that ponies with low antibody levels exhibited partial clinical protection when rechallenged up to 18 months following first infection, suggesting that immune mechanisms involving rapid stimulation of both B and T cells responses could assist in protection [15,16]. Studies carried out using interferon gamma (IFN-) protein synthesis as a marker for a cell mediated immune (CMI) response have demonstrated an increase in IFN- gene expression following vaccination with a canarypox recombinant [17,18], an Immuno Stimulating Complex (ISCOM) subunit [19], an ISCOM-matrix subunit [20] and an inactivated whole virus vaccine [21]. In this study real time RT-PCR was used to quantify cytokine gene expression. This method has been validated in the profiling of human and equine cytokines [22,23] but post transcriptional and post translational modifications may result in imperfect correlation between mRNA and protein synthesis. However a strong correlation between mRNA and secreted protein levels has been demonstrated for human IFN- following vaccination [24]. The objective of this study was to extend our previous comparative vaccine study in Thoroughbred horses and evaluate both the humoral antibody response and antiviral cytokine expression following vaccination with a whole virus (Duvaxyn IE-T Plus), a subunit ISCOM-matrix (Equilis Prequenza Te) and a canarypox recombinant vaccine (ProteqFlu-Te).

2. Material and methods 2.1. Horses This study was carried out in a population of 44 unvaccinated Thoroughbred weanlings on a private stud farm. The weanlings ranged in age from 183 to 342 days (mean 259 ± 6.8 SE days) at the time of first vaccination (V1). 2.2. Vaccines The whole virus vaccine Duvaxyn IE-T Plus (Elanco Animal Health), ISCOM-matrix subunit vaccine Equilis Prequenza Te (MSD Animal Health) and canarypox recombinant vaccine ProteqFlu-Te (Merial) included in this study were purchased commercially. The composition of these vaccines has previously been described [13]. 2.3. Vaccination The weanlings were randomly allocated one of the three vaccines and received two doses (V1 and V2) 29 days apart by deep intramuscular injection. Fourteen weanlings received Duvaxyn IE-T

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Plus, 15 received Equilis Prequenza Te and 15 received ProteqFluTe. 2.4. Collection of samples Whole blood and PAXgene blood samples (PreAnalytix, Switzerland) were collected at V1, two, seven and 14 days post V1. Additional whole blood samples were collected at V2 and 21 days post V2. 2.5. Serology Antibodies against A/eq/Newmarket/2/93 (H3N8), A/eq/Meath/07 (H3N8), A/eq/South Africa/4/03 (H3N8) and A/eq/Prague/56 (H7N7) were measured using the SRH test as previously described [13]. Seroconversion was defined as an increase in SRH antibody level of 25 mm2 or 50% whichever was smaller between the paired serum samples [11]. A poor responder was defined as a horse that did not mount a mean H3N8 SRH antibody response of >25 mm2 post vaccination [13].The laboratory investigator was blinded to vaccine allocation to individual horses. 2.6. Relative quantification of cytokine gene expression using real-time RT-PCR PAXgene blood samples were processed as per the manufacturer’s instructions and extracted RNA was quantified by measuring absorbance at 260 nm. Reverse transcription and quantitative realtime PCR reactions were carried out using the AgPath-ID One Step RT-PCR Kit (Ambion/Applied Biosystems, Foster City, CA, USA) on an ABI Taqman 7500 platform. Primer probe sets for the detection of IL-1␤ [25], IL-4, IFN- [18] and an endogenous control, ␤-GUS [25] were used as previously described. ␤-GUS is consistently expressed in experimental samples, does not have processed pseudogenes and is commonly used in equine cytokine expression studies [18,25–27]. Reverse transcription reactions were carried out using half a microgram of total cellular RNA. For IL-1␤, each 25 ␮l reaction contained 5 ␮l RNA, 2× RT-PCR buffer, 80 ng tRNA (Sigma–Aldrich), 0.6 ␮M of each primer, 0.3 ␮M probe and 25× RT-PCR enzyme. For IL-4, IFN-, and ␤-GUS primer concentrations were 0.2 ␮M, 0.2 ␮M and 0.4 ␮M respectively. Each sample was tested in triplicate for each of the cytokine targets and the endogenous control. One step RT- PCR was carried out at 45 ◦ C for 10 min followed by 95 ◦ C for 10 min, 45 cycles of 95 ◦ C for 15 s and 60 ◦ C for 60 s. Data were analysed as a relative quantification study with day 0 samples as the calibrator for each horse. Relative quantification was then used to compare gene expression levels post vaccination using the 2−CT method [28]. Results are shown as the mean (±SEM) fold changes in cytokine gene expression between the different vaccine groups. 2.7. Statistical analysis The area under the curve (AUC) as described by Heldens et al., [29] was calculated by the trapezoidal rule and used as the metric for the repeated measures analysis of antibody levels. IBM SPSS Statistics 21 (Armonk, New York, USA) was used to analyse the data. Repeated measures analysis of variance and post hoc testing was carried out using the Kruskal–Wallis and the Mann–Whitney U tests. Test of significance were carried out at ˛ = 5% level.

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3. Results 3.1. Pattern of humoral antibody response post vaccination The H3N8 pattern of humoral antibody response was similar for all vaccines (Figs. 1 and 2, Supplementary Fig. 1). Overall horses responded poorly to V1 (mean SRH 8.67 ± 4.410 SE mm2 and 72.23 ± 10.962 SE mm2 seven and 14 days post V1 respectively) but mounted a better response three weeks post V2 (mean SRH 188.33 ± 6.948 SE mm2 ). Three of the weanlings (21%) vaccinated with Duvaxyn IE-T Plus and one of the weanlings (7%) vaccinated with Equilis Prequenza Te seroconverted (H3N8) one week after V1. Post V1 the H3N8 antibody levels of weanlings vaccinated with Duvaxyn IE-T Plus and Equilis Prequenza Te peaked at 14 days (mean SRH 145.21 ± 12.305 SE mm2 and 39.30 ± 16.559 SE mm2 respectively). Post V1 the H3N8 antibody levels of weanlings vaccinated with ProteqFlu-Te peaked at 29 days (mean SRH 44.23 ± 11.677 SE mm2 ). No SRH antibody response against H7N7 was detected in weanlings vaccinated with the canarypox recombinant vaccine ProteqFlu-Te which contains H3N8 virus only (Supplementary Fig. 2). The H7N7 antibody response of weanlings vaccinated with Duvaxyn IE-T Plus and Equilis Prequenza Te were similar (Supplementary Fig. 2). Furthermore, the H7N7 antibody levels for both vaccine groups was similar (<25 mm2 difference) to that of their H3N8 antibody levels at each time point following V1 and V2.

3.2. Comparison of humoral antibody response post vaccination Following V1 the antibody response of the horses vaccinated with Duvaxyn IE-T Plus against all three H3N8 antigens was significantly higher than that of the horses vaccinated with the other two products (p < 0.001). There was no significant difference in H3N8 antibody response following V1 between weanlings vaccinated with Equilis Prequenza Te and ProteqFlu-Te. Three weeks post V2 the antibody response of horses vaccinated with Duvaxyn IE-T Plus against A/eq/Meath/07 was significantly higher than those vaccinated with the other two products (p = 0.009). Three weeks post V2 the antibody response of horses vaccinated with Duvaxyn IE-T Plus against A/eq/South Africa/4/03 was significantly higher than those vaccinated with ProteqFlu-Te (p = 0.014) but not significantly higher than those vaccinated with Equilis Prequenza Te. Three weeks post V2 there was no significant difference in antibody response against A/eq/Newmarket/2/93 between the three vaccine groups. Following V1, the H7N7 antibody response of horses vaccinated with Duvaxyn IE-T Plus was significantly higher than that of horses vaccinated with Equilis Prequenza Te (p = 0.001). Following V2 there was no significant difference in H7N7 antibody response between the two vaccine groups.

3.3. Area under the curve (AUC) The AUC of the SRH levels of the weanlings vaccinated with Duvaxyn IE-T Plus against all three H3N8 antigens was significantly greater than that of the weanlings vaccinated with the two other products (p < 0.001). There was no significant difference in SRH AUC between weanlings vaccinated with Equilis Prequenza Te compared with those vaccinated with ProteqFlu-Te. The AUC of the H7N7 SRH levels of the weanlings vaccinated with Duvaxyn IE-T Plus was significantly greater than that of the weanlings vaccinated with Equilis Prequenza Te (p = 0.002).

Table 1 Percentage of poor responders to H3N8 in each vaccine group. Vaccine

Post V1

Duvaxyn IE-T Plus Equilis Prequenza Te ProteqFlu-Te

0% (0/14) 67% (10/15) 47% (7/15)

Post V2 0% (0/14) 7% (1/15) 0% (0/15)

3.4. Failure or delay in humoral antibody response to vaccination Failure to seroconvert to H3N8 (poor responders) was examined post V1 and V2 (Table 1). One horse vaccinated with Equilis Prequenza Te failed to seroconvert following V1 and V2. A second horse vaccinated with Equilis Prequenza Te seroconverted following V1 but failed to seroconvert following V2. This horse had a mean H3N8 antibody level of 181.26 mm2 at the time of V2. A delayed response to V1 was observed in two horses vaccinated with ProteqFlu-Te. These horses failed to respond two weeks post V1, but responded two weeks later, i.e. four weeks post V1. 3.5. Cellular immune response post vaccination Vaccination with all three vaccines resulted in a significant (p < 0.01) increase in IFN- gene expression which peaked at 7 days post V1 (Fig. 3). There was no significant difference in IFN- gene expression between horses vaccinated with the whole inactivated, the subunit and the canarypox recombinant vaccine included in this study. Mean gene expression levels of IL-1␤ and IL-4 peaked on day 14 post V1 (Figs. 4 and 5). IL-1␤ gene expression of horses vaccinated with Duvaxyn IE-T Plus was greater than that observed by horses vaccinated with the other two products however the difference between the vaccine groups was not statistically significant. On day 14 post V1, IL-4 gene expression levels in weanlings vaccinated with Equilis Prequenza Te were significantly greater (p = 0.012) than in those vaccinated with Duvaxyn IE-T Plus and ProteqFlu-Te. The number of horses in each vaccine group which experienced a two fold increase or greater in gene expression level (IFN-, IL-1␤, IL-4) post V1 is shown in Supplementary Table 1. 4. Discussion On the basis of H3N8 and H7N7 SRH antibody levels the results of this study demonstrate that the whole virus vaccine Duvaxyn IE-T Plus was superior to the other products tested. The findings of this study are consistent with those previously reported by our laboratory [13]. It is possible that the combined adjuvant formulation of aluminium hydroxide and Carbomer (Carbopol 934P) may play a role in stimulating a superior antibody response compared with vaccines which contain the single adjuvants ISCOM-matrix (Equilis Prequenza Te) and Carbomer only (ProteqFlu-Te). The updating of a combined EIV and equine herpes virus vaccine to include a second adjuvant resulted in a significant increase in immunogenicity [30] when compared with the original vaccine [31]. A comparison of antibody response stimulated following vaccination with Duvaxyn IE-T Plus and the same vaccine formulation without the tetanus component (Duvaxyn IE Plus) which contains a single adjuvant (Carbopol 934P) would assist in the elucidation of the benefit of the inclusion of aluminium hydroxide. The pattern of antibody response was similar for all vaccines. Three of the weanlings vaccinated with Duvaxyn IE-T Plus and one of the weanlings vaccinated with Equilis Prequenza Te seroconverted (H3N8) one week after V1. This is consistent with previous reports [32–34]. Detectable SRH antibody levels have also been reported seven days following V1 with a canarypox recombinant vaccine in Welsh mountain ponies [35]. However, in this study none of the weanlings vaccinated with ProteqFlu-Te had measurable SRH

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Fig. 1. Mean SRH antibody response against A/eq/Meath/07 (H3N8). Broken lines = SRH antibody level 85 mm2 and 150 mm2 correlating with clinical and virological protection respectively, error bars represent standard error of the mean.

Fig. 2. Mean SRH antibody response against A/eq/South Africa/4/03 (H3N8). Broken lines = SRH antibody level 85 mm2 and 150 mm2 correlating with clinical and virological protection respectively, error bars represent standard error of the mean.

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Fig. 3. Relative quantification of IFN- mRNA by RT-PCR post V1. Mean fold change levels ± SEM in gene expression. RQ = Relative quantification.

Fig. 4. Relative quantification of IL-1␤ mRNA by RT-PCR post V1. Mean fold change levels ± SEM in gene expression. RQ = Relative quantification.

antibodies (H3N8) until at least 14 days post V1 and their antibodies peaked later than the other vaccine groups. A delay in response to vaccination has previously been reported with this product in a population of National Hunt horses [36]. Evidence of the stimulation of a CMI response following vaccination with ISCOM based vaccines has previously been demonstrated in mice, humans and in horses [19,37,38]. The stimulation of a CMI response following vaccination with a canarypox recombinant vaccine has also been reported in humans following vaccination against HIV [39,40] and in horses following vaccination against EIV

and West Nile Virus [17,18,41]. Post V1 an increase in IFN- has been reported after 14 and 35 days with a canarypox recombinant vaccine [17,18] and after 21 days with a subunit ISCOM-matrix based vaccine [20]. In 1994, the findings of a study by Hannant et al., suggested that whole inactivated virus vaccines failed to induce EIV-specific cytotoxic T lymphocyte activity [42]. However, a recent study by Paillot et al., demonstrated an increase in IFN- in experimental ponies 21 and 13 days post V1 and V2 respectively with the whole virus vaccine [21]. In the current study vaccination with all three vaccines resulted in a significant increase

Fig. 5. Relative quantification of IL-4 mRNA by RT-PCR post V1. Mean fold change levels ± SEM in gene expression. RQ = Relative quantification; * = IL-4 gene expression levels in weanlings vaccinated with Equilis Prequenza Te significantly greater than in those vaccinated with Duvaxyn IE-T Plus and ProteqFlu–Te.

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in IFN- gene expression which peaked at 7 days post vaccination. To the best of our knowledge, this is the first report of a significant increase in IFN- gene expression within one week post V1 with these vaccines. Overall, there was no significant difference in IFN- gene expression following vaccination with the whole virus, ISCOM subunit or canarypox recombinant vaccines included in this study. Cellular immune responses to influenza are largely directed against the conserved internal influenza virus proteins NP, M1, PB1 and PB2 [43–46]. Irrespective of the more modern ISCOM-matrix and canarypox recombinant vaccine presentation systems which are designed to stimulate both a B and T cell immune response, the vaccines in this study contain only surface antigens which stimulate a humoral antibody response and are unlikely to have major T cell epitopes. Future subunit and recombinant vaccines might be improved by the inclusion of proteins that stimulate a strong CMI response. The difference in IL-1␤ gene expression between the vaccine groups was not statistically significant. Mean gene expression levels of IL-4 peaked on day 14 post V1 and were greatest in weanlings vaccinated with the subunit ISCOM-matrix based vaccine. An increase in IL-4 following vaccination with ISCOM based vaccines has previously been reported in other animal models [47–49]. The findings of the present study which reported no increase in IL-4 gene expression following vaccination with a canarypox recombinant vaccine are in agreement with those of Adams et al., [18]. In a study carried out by Onmaz et al., an increase in IL-4 activity was demonstrated as early as four hours post V1 with Duvaxyn IE-T [50]. In this study, weanlings vaccinated with the same product demonstrated an increase in IL-4 gene expression; however this was not statistically significant. The earliest sample collection in the current study was two days post V1. Further investigations at alternative sampling points may be beneficial. The absence of poor responders in weanlings vaccinated with Duvaxyn IE-T Plus is in agreement with our previous study, as is the incidence of poor responders to vaccination with the canarypox recombinant vaccine [13]. The incidence of poor responders to Equilis Prequenza Te is higher than that previously identified. The reason why some horses fail to mount an adequate response to vaccination remains to be elucidated but genetic variation in response to influenza vaccination in humans has been reported [51,52]. The absence of poor responders to vaccination among weanlings vaccinated with the whole virus vaccine may be due to the fact that such vaccines are structurally more complex than subunit vaccines and offer more opportunity to avoid genetic restrictions [53]. In this study the CMI response of weanlings which failed to mount a significant antibody response to vaccination was comparable with normal responders thus suggesting that failure to respond was limited to humoral response only (data not shown). In conclusion, this is the first study to carry out a comparison of both humoral antibody and CMI responses induced following primary vaccination with a selection of commercially available products. On the basis of SRH antibody levels the whole virus vaccine Duvaxyn IE-T Plus was superior to the other products tested. Using IFN- gene expression as a marker for cell mediated immunity, this present study demonstrated there was no significant difference in IFN- gene expression stimulation by the whole inactivated, the subunit and the canary-pox recombinant vaccines.

Acknowledgements This study would not have been possible without the cooperation of the veterinary surgeon, owner and animal handlers to whom the authors are extremely grateful. All of the experimental work was funded by the Department of Agriculture under the National Development Plan and carried out at the Irish Equine

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Centre. Statistical analysis was carried out by Dr. Jean Saunders at the Statistical Consulting Unit at the University of Limerick. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine. 2013.08.083. References [1] Webster RG. Are equine 1 influenza viruses still present in horses? Equine Vet J 1993;25:537–8. [2] OIE Bulletin. OIE Expert surveillance panel of equine influenza vaccines–conclusions and recommendations. Mill Hill, London (United Kingdom) 18th January, 2008 (2) 42–5. [3] Cullinane A. Equine influenza – a constantly evolving challenge. Equine Vet Educ 2009;8:1–7. [4] Guthrie AJ, Stevens KB, Bosman PP. The circumstances surrounding the outbreak and spread of equine influenza in South Africa. Rev Sci Tech 1999;18(1):179–85. [5] King EL, Macdonald D. Report of the Board of Inquiry appointed by the Board of the National Horseracing Authority to conduct enquiry into the causes of the equine influenza which started in the Western cape in early December 2003 and spread to the Eastern Cape and Gauteng. Aust Equine Vet 2004;23:139–42. [6] Uppal PK, Yadav MP, Oberoi MS. Isolation of A/Equi-2 virus during 1987 equine influenza epidemic in India. Equine Vet J 1989;21(5):364–6. [7] Powell DG, Watkins KL, Li PH, Shortridge KF. Outbreak of equine influenza among horses in Hong Kong during. Vet Rec 1995;136(21):531–6. [8] Garner MG, Cowled B, East IJ, Moloney BJ, Kung N. Evaluating the effectiveness of the response to equine influenza in the Australian outbreak and the potential role of early vaccination. Aust Vet J 2011;89(Suppl. 1):143–5. [9] Mumford JA, Wood J. Establishing an acceptability threshold for equine influenza vaccines. Dev Biol Stand 1992;79:137–46. [10] Mumford JA, Jessett D, Dunleavy U, Wood J, Hannant D, Sundquist B, et al. Antigenicity and immunogenicity of experimental equine influenza ISCOM vaccines. Vaccine 1994;12(9):857–63. [11] Newton JR, Townsend HG, Wood JL, Sinclair R, Hannant D, Mumford JA. Immunity to equine influenza: relationship of vaccine-induced antibody in young Thoroughbred racehorses to protection against field infection with influenza A/equine-2 viruses (H3N8). Equine Vet J 2000;32(1):65–74. [12] Mumford JA. Biology epidemiology and vaccinology of equine influenza. Quality control of equine influenza vaccines. In: Proceedings of an International Symposium organised by the European Directorate for the Quality of Medicines (EDQM). 2001. p. 7–12. [13] Gildea S, Arkins S, Walsh C, Cullinane A. A comparison of antibody responses to commercial equine influenza vaccines following primary vaccination of Thoroughbred weanlings-a randomised blind study. Vaccine 2011;29(49):9214–23. [14] Townsend HG, Penner SJ, Watts TC, Cook A, Bogdan J, Haines DM, et al. Efficacy of a cold-adapted, intranasal, equine influenza vaccine: challenge trials. Equine Vet J 2001;33(7):637–43. [15] Hannant D, Mumford JA, Jessett DM. Duration of circulating antibody and immunity following infection with equine influenza virus. Vet Rec 1988;122(6):125–8. [16] Bryant NA, Paillot R, Rash AS, Medcalf E, Montesso F, Ross J, et al. Comparison of two modern vaccines and previous influenza infection against challenge with an equine influenza virus from the Australian 2007 outbreak. Vet Res 2010;41(2):19. [17] Paillot R, Kydd JH, MacRae S, Minke JM, Hannant D, Daly JM. New assays to measure equine influenza virus-specific Type 1 immunity in horses. Vaccine 2007;25(42):7385–98, 16. [18] Adams AA, Sturgill TL, Breathnach CC, Chambers TM, Siger L, Minke JM, et al. Humoral and cell-mediated immune responses of old horses following recombinant canarypox virus vaccination and subsequent challenge infection. Vet Immunol Immunopathol 2011;139(2–4):128–40. [19] Paillot R, Grimmett H, Elton D, Daly JM. Protection, systemic IFNgamma, and antibody responses induced by an ISCOM-based vaccine against a recent equine influenza virus in its natural host. Vet Res 2008;39(3):21. [20] Paillot R, Prowse L. ISCOM-matrix-based equine influenza (EIV) vaccine stimulates cell-mediated immunity in the horse. Vet Immunol Immunopathol 2012;145(1–2):516–21. [21] Paillot R, Prowse L, Montesso F, Huang CM, Barnes H, Escala J. Whole inactivated equine influenza vaccine: efficacy against a representative clade 2 equine influenza virus, IFNgamma synthesis and duration of humoral immunity. Vet Microbiol 2013;162(2–4):396–407. [22] Stordeur P, Zhou L, Byl B, Brohet F, Burny W, de Groote D, et al. Immune monitoring in whole blood using real-time PCR. J Immunol Methods 2003;276(1–2):69–77. [23] Breathnach CC, Rudersdorf R, Lunn DP. Use of recombinant modified vaccinia Ankara viral vectors for equine influenza vaccination. Vet Immunol Immunopathol 2004;98(3–4):127–36. [24] Shebl FM, Pinto LA, Garcia-Pineres A, Lempicki R, Williams M, Harro C, et al. Comparison of mRNA and protein measures of cytokines following

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