Antibody and cellular immune responses following DNA vaccination and EHV-1 infection of ponies

Veterinary Immunology and Immunopathology 111 (2006) 81–95 www.elsevier.com/locate/vetimm

Antibody and cellular immune responses following DNA vaccination and EHV-1 infection of ponies G. Soboll a, S.B. Hussey a, J.M. Whalley b, G.P. Allen c, M.T. Koen b, N. Santucci b, D.G. Fraser d, M.D. Macklin e, W.F. Swain e, D.P. Lunn a,* a

Department of Clinical Sciences, College of Veterinary Medicine, Colorado State University, 300W. Drake Rd., Fort Collins, Colorado 80523, USA b Department of Biological Sciences, Macquarie University, Sydney, Australia c Department of Veterinary Sciences, University of Kentucky Lexington, KY 40546, USA d Department of Veterinary Microbiology and Immunology, Washington State University, Pullman, WA 99164, USA e PowderJect Vaccines Inc., 585 Science Drive, Suite C, Madison, WI 53711, USA

Abstract Equine herpesvirus-1 (EHV-1) is the cause of serious disease with high economic impact on the horse industry, as outbreaks of EHV-1 disease occur every year despite the frequent use of vaccines. Cytotoxic T-lymphocytes (CTLs) are important for protection from primary and reactivating latent EHV-1 infection. DNA vaccination is a powerful technique for stimulating CTLs, and the aim of this study was to assess antibody and cellular immune responses and protection resulting from DNA vaccination of ponies with combinations of EHV-1 genes. Fifteen ponies were divided into three groups of five ponies each. Two vaccination groups were DNA vaccinated on four different occasions with combinations of plasmids encoding the gB, gC, and gD glycoproteins or plasmids encoding the immediate early (IE) and early proteins (UL5) of EHV-1, using the PowderJect XR research device. Total dose of DNA/plasmid/vaccination were 25 mg. A third group comprised unvaccinated control ponies. All ponies were challenge infected with EHV-1 6 weeks after the last vaccination, and protection from clinical disease, viral shedding, and viremia was determined. Virus neutralizing antibodies and isotype specific antibody responses against whole EHV-1 did not increase in either vaccination group in response to vaccination. However, glycoprotein gene vaccinated ponies showed gD and gC specific antibody responses. Vaccination did not affect EHV-1 specific lymphoproliferative or CTL responses. Following challenge infection with EHV-1, ponies in all three groups showed clinical signs of disease. EHV-1 specific CTLs, proliferative responses, and antibody responses increased significantly in all three groups following challenge infection. In summary, particle-mediated EHV-1 DNA vaccination induced limited immune responses and protection. Future vaccination strategies must focus on generating stronger CTL responses. # 2006 Elsevier B.V. All rights reserved. Keywords: EHV-1; Horses; DNA vaccination; CTL

* Corresponding author. Tel.: +1 970 297 1274; fax: +1 970 297 1275. E-mail address: [email protected] (D.P. Lunn). 0165-2427/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2006.01.011

82

G. Soboll et al. / Veterinary Immunology and Immunopathology 111 (2006) 81–95

1. Introduction Equine herpesvirus-1 (EHV-1) and -4 (EHV-4) are the cause of serious disease that is ubiquitous in horses all over the world (Bryans and Allen, 1989). While EHV-4 is more prevalent (Edington et al., 1994; Gilkerson et al., 1999b), EHV-1 typically causes more severe disease (Allen et al., 1999). Infection with both viruses results in respiratory disease, and EHV-1 is the cause of abortion storms, neonatal foal death, and myeloencephalopathy leading to significant economic losses in the horse industry. The high prevalence of EHV-1 and -4 related diseases result from a high rate of initial respiratory infection in young horses, the establishment of latency in lymphocytes and neuronal tissues, and frequent episodes of viral reactivation during which silent carriers transmit the virus to new equine hosts (Allen et al., 1999). While horses with repeated exposure to the virus become resistant to the clinical form of the respiratory disease (Kydd et al., 1994a,b), viremia resulting from recrudescing infections or superinfections with EHV-1 is the cause of devastating abortion storms and sporadic neuropathies. Therefore, a primary goal for protection from EHV1 abortion has to be the control of viremia. Following infection with EHV-1 there is a short period of post-infection immunity lasting 3–6 months (Bryans and Allen, 1989; Doll et al., 1955). Research efforts have focused on identifying the immune mechanisms that lead to this convalescent immunity. The role of virus neutralizing (VN) antibodies may be restricted to the clearance of limited amounts extracellular progeny virus from the bloodstream (van Der Meulen et al., 2000). VN titers have therefore been unsatisfactory as a correlate for protection from viremia and abortion (Burki et al., 1990; Mumford et al., 1987). In contrast, CD8+ MHC class I-restricted cytotoxic T-lymphocytes (CTLs) are believed to play a major role in clearance of viremic lymphocytes (Allen et al., 1999, 1995). Limiting dilution analysis studies have shown that the frequencies of EHV-1 specific CTL precursors are correlated with protection from EHV-1 infection (Kydd et al., 2003; O’Neill et al., 1999). Currently available vaccines for EHV-1 include killed virus adjuvanted vaccines, and modified live virus vaccines. Killed virus vaccines typically induce

only circulating antibody responses, which are inefficient in controlling infection (Burki et al., 1990). This is because the proteins contained in inactivated vaccines are not typically processed by the MHC I pathway, which is necessary for the induction of CTLs (Audibert and Lise, 1993). Modified live vaccines for EHV-1 are capable of inducing antibody and CTL responses (Ellis et al., 1995), but are not recommended for use in pregnant animals which are the primary candidates in need of vaccination (Allen et al., 1999; Oehen et al., 1991). DNA vaccination is an attractive strategy for prevention of EHV-1 infection, because it induces both potent CTL and antibody responses, safety concerns are minimal, and production of DNA vaccines is relatively easy and inexpensive (Cohen, 1993; Hassett and Whitton, 1996). The use of DNA vaccination for EHV-1 has shown promising results in mice (Ruitenberg et al., 2000b, 1999a,b; Walker et al., 1997) and in the horse (Ruitenberg et al., 2000a). In addition, we have previously demonstrated the efficacy of DNA vaccination in ponies for protection from equine influenza virus infection (Lunn et al., 1999; Soboll et al., 2003a,b). DNA vaccination is thought to be well suited for EHV-1 vaccination because it directly targets dendritic cells, which are professional antigen presenting cells (Banchereau and Steinman, 1998; Rouse et al., 1994) and have previously been shown to be extremely efficient at inducing and stimulating CTLs (Bhardwaj et al., 1995; Ludewig et al., 1998). For the development of a DNA vaccine for EHV-1 in the horse, it is important to know which components of EHV-1 must be processed and presented by dendritic cells to stimulate the appropriate immune responses in vivo. This means that genes encoding proteins containing CTL epitopes must be used. It has been proposed that viral proteins presented early in the replication cycle, such as the immediate early (IE) and early (UL5) gene for EHV, are likely to be important CTL targets (Koen et al., 2000; Ruitenberg et al., 1999b; Siedek et al., 1999; Smith et al., 1998). Recently we showed that in ponies carrying the A 3.1 genotype the IE protein contains CTL epitopes (Soboll et al., 2003a,b). In murine studies, the glycoproteins of herpesviruses have been identified as containing CTL epitopes (Gallichan and Rosenthal, 1996), and several studies in mice have reported protection from EHV-1

G. Soboll et al. / Veterinary Immunology and Immunopathology 111 (2006) 81–95

by vaccination with gB, gC, gD, gH, and gL glycoproteins (Allen and Yeargan, 1987; Crabb et al., 1991; Flowers et al., 1995; Kukreja et al., 1998; Osterrieder et al., 1995; Packiarajah et al., 1998; Stokes et al., 1996; Wellington et al., 1996a,b; Whalley et al., 1995). However, protection associated with immune responses to these EHV-1 components has not been evaluated in horses. The aim of this study was to test the value of particle-mediated DNA immunization of ponies using either a combination of the IE and UL5 genes, or a combination of the gB/gC/gD genes for inducing protection from EHV-1 infection. Specifically we wanted to examine EHV-1 specific lymphoproliferative responses, MHC I restricted CTL responses, VN antibodies, and circulating and mucosal antibody isotype responses. A secondary goal of this study was to establish a respiratory infection model and perform a comprehensive examination of the events following infection. This examination included testing for latent infection, clinical evaluation, examination of nasal virus shedding, and the determination of lymphocyte associated viremia.

2. Material and methods 2.1. Experimental animals Fifteen ponies were used for this study. The ponies were approximately 1 year of age and of both sexes. This age was chosen in the hopes to find animals that had not yet been latently infected. During the study ponies were each housed in individual isolation stalls. The ponies were fed twice a day with a diet of hay and pelleted concentrate. The maintenance and experimental protocols followed the animal care guidelines of the Research and Animal Resources Committee, University of Wisconsin. 2.2. Equine herpesvirus virus preparation and isolation This experiment used equine herpes virus 1, strain Army 183 (EHV-1/A183). For experimental infections and in vitro assays, the virus was propagated in equine dermal cells (ATCC CCL-57) and used as virus infected tissue culture fluid.

83

2.3. Preparation of EHV-1 encoding plasmids for use in DNA vaccines The EHV-1 glycoproteins (gB, gC, and gD), the immediate early protein, and the UL5 early protein have been characterized extensively (Allen and Yeargan, 1987; Crabb et al., 1991; Flowers et al., 1995; Koen et al., 2000; Osterrieder et al., 1995; Stokes et al., 1996; Tewari et al., 1994; Wellington et al., 1996a,b; Whalley et al., 1995). Genes encoding EHV-1 glycoproteins (gC and gD), the immediate early protein and the UL5 early protein were cloned into the pCR3.1 plasmid (Invitrogen Inc., Carlsbad, CA). The gB glycoprotein was cloned into the pRc/ CMV plasmid (Invitrogen) (Munro et al., 1999). These vectors contained the ampicillin resistance gene and the immediate early promoter and intron-A of human cytomegalovirus. For use in the DNA vaccines, the plasmids were purified by anion exchange resin chromatography (Qiagen Inc., Chatsworth, CA). For preparation of the particle-mediated vaccines, gB/gC/gD DNA or IE/UL5 and empty vector DNA were mixed and condensed onto gold particles at 0.83 mg of each DNA per mg of gold. The coated gold particles were distributed onto the inner surface of TefzelTM tubing at 0.5 mg gold particles per 0.5-in. tubing length. Subsequently, 0.5-in. sections of tubing were loaded into the PowderJect1 XR-1 research device. Each discharge therefore delivered 0.42 mg of each DNA. 2.4. Experimental design Fifteen ponies were assigned to three groups of five ponies, and designated ‘‘gB/gC/gD DNA vaccination group’’, ‘‘IE/UL5 DNA vaccination group’’ and ‘‘control group’’. Ponies in the DNA vaccination groups were vaccinated on Days 0, 41, 86, and 126 at skin and mucosal sites using the PowderJect1 XR-1 research device. Ponies in the gB/gC/gD DNA vaccination group received gB, gC, and gD DNA and ponies in the IE/UL5 DNA vaccination group received IE, UL5, and empty vector DNA. Empty vector DNA was included to equalize the amount of EHV-1 plasmid in the vaccines. For particle-mediated DNA vaccinations, ponies were sedated with xylazine HCl (1 mg/kg, IV; Miles, Shawnee, KS) and briefly anesthetized with ketamine (2.2 mg/kg, IV; Ketaset,

84

G. Soboll et al. / Veterinary Immunology and Immunopathology 111 (2006) 81–95

Fort Dodge, IA). Ponies were vaccinated at 24 inguinal skin sites, 8 perineal skin sites, 24 ventral tongue sites, and 4 sites of the conjunctiva and third eyelid. These target sites were chosen based on previous experiments performed in our laboratory that showed when ponies were vaccinated at skin and mucosal sites, the vaccine efficacy was improved when compared to ponies that were vaccinated at skin sites only (Lunn et al., 1999). Total dose of each EHV1 gene was 25 mg of DNA per vaccination. One control pony was kept throughout the duration of the study as a sentinel for EHV-1 infection. The other four control ponies were acquired 3 weeks prior to challenge infection. All fifteen ponies were challenge infected on Day 153 by intranasal instillation of 2
107 PFU of EHV-1/A183 per pony in a volume of 8 ml tissue culture supernatant using a five 1/4 in. 14 gauge catheter (MILA Inc., Covington, KY). Ponies were studied for 28 days following the challenge infection. 2.5. Sample collection Serum and nasal secretions for antibody assays were sampled at bi-weekly intervals prior challenge infection and twice weekly for 3 weeks following challenge infection. Blood for preparation of serum was collected by jugular venipuncture into serum separator tubes (Becton Dickinson Company, Franklin Lake, NJ). Nasal secretions were collected by placing a cotton tampon in the ventral nasal meatus for at least 15 min, and subsequent centrifugation at 2000
g. Typical recovery of nasal secretions was between 0.5 and 1.5 ml. Narrow tampons (Tampax slender regular, Procter & Gamble, Cincinnati, OH) were used to ensure, that no hemorrhage occurred from the sampling procedure. The ponies were sedated before sampling with 20–40 mg/kg Detomidine by IV injection (Pfizer Animal Health Inc., West Chester, PA). Serum and nasal secretion samples were aliquoted and stored at 20 8C. Physical examinations, blood for detection of viremia, and nasal swab samples for virus isolation were collected daily for 10 days and every other day from Day 10 to Day 21 after challenge infection. Nasal swabs were collected using Dacron swabs (Baxter Healthcare Corporation, McGaw Park, IL). Swabs were stored in 1 ml of virus transport medium at 70 8C. Blood for detection of viremia was collected by jugular

venipuncture into heparinized tubes and processed as described in Section 2.9 below. For cryopreservation of PBMCs to use in CTL assays, lymphoproliferative assays, MHC typing, and isolation of DNA, blood was collected into heparin by jugular venipuncture. 2.6. Detection of latent EHV-1 infection in lymphocytes For detection of latent EHV-1 infection, 107 PBMCs from each pony were isolated over Histopaque-1077 (Sigma, St. Louis, MO). Cellular nucleic acid was isolated using the High-Pure Template Preparation Kit (Roche, Diagnostics Corporation, Indianapolis, IN). Latently infected ponies were identified using a nested PCR, as described previously (Borchers and Slater, 1993). 2.7. MHC class I typing Ponies were typed for their major histocompatibility complex (MHC) class I molecules, encoded by the ELA-A locus, by PBMC microcytotoxicity assays using previously described antisera (Bailey et al., 1984, 2000). The equine ELA-A locus includes the internationally accepted alleles, A1–A10, A14, A15, and A19 and provisionally accepted alleles, W16, W17, W18, and W20 (Antczak, 1992; Bailey et al., 2000). Available antisera at the time of typing included those for A1–A10 as well as those for the less well-characterized W11 specificity. 2.8. Detection of viral shedding Viral shedding in nasal swab samples was measured in PFU/ml of sample. For this purpose equine dermal cells were added to six-well tissue culture plates (Corning Inc., Corning, NY) and allowed to adhere. When cell layers were 90% confluent, nasal swab samples were thawed and filter sterilized using a 0.45 mM syringe top filter (Pall Corp., Ann Arbor, MI). Filtered samples were added to equine dermal cell layers at 10-fold dilutions in duplicate and incubated at 37 8C, 4% CO2 for 1.5 h. Inoculums were then removed, plates were rinsed with minimal essential media MEM (GibcoBRL, Life Technologies Inc., Grand Island, NY) plus 2% fetal calf serum and 3 ml of methylcellulose media (MEM

G. Soboll et al. / Veterinary Immunology and Immunopathology 111 (2006) 81–95

containing 0.75% methyl cellulose (Sigma), 100 U ml1 penicillin, 100 mg/ml streptomycin, NaHCO3, amino acids, vitamins, glutamine, amphotericin, and 2% fetal calf serum,) was added to each well. Plates were incubated for 3 days at 37 8C, 4% CO2, before removal of the methylcellulose media and staining with 1 ml of crystal violet (Fisher Scientific, Fair Lawn, NJ) per well for 30 min. After rinsing with water and drying, plaques were counted and the PFU/ ml of sample was calculated. 2.9. Viremia For detection of viremia, 15 ml of blood was collected into heparin and red blood cells were allowed to settle. The plasma, containing PBMCs, was collected, white blood cells were pelleted, and washed three times in PBS and subsequently added to 25 cm2 tissue culture flasks (Corning Inc.) containing a 90% confluent monolayer of equine dermal cells. Flasks were incubated for 7 days at 37 8C, 4% CO2 and checked daily for appearance of cytopathic effect (CPE). After 7 days, the virus containing supernatants were recovered by freeze thawing and 500 ml aliquots were added to a fresh 25 cm2 tissue culture flasks containing a 90% confluent monolayer of equine dermal cells. These second pass flasks were incubated for further 7 days at 37 8C, 4% CO2 and appearance of cytopathic effect was recorded as a positive result. 2.10. Antibody immunoassays Virus neutralizing antibody titers in serum sample were determined at the Wisconsin State Veterinary Diagnostic Laboratory (Madison, WI). Briefly, serial dilutions of each serum sample from 1:2 to 1:4096 was added to 96-well tissue culture plates (Corning Inc.) and incubated with EHV-1 at 37 8C, 5% CO2 for 1 h. Subsequently Madin-Darbey bovine kidney cells were added and plates were incubated for 4 further days at 37 8C, 5% CO2, before determination of CPE. Each sample group included EHV-1 positive and negative sera as controls. Serum and nasal secretions were also examined for EHV-1/A183—specific IgG isotype and IgA antibodies using an ELISA system. For this purpose 10 mg/ml of concentrated EHV-1 in carbonate-coating buffer was coated onto 96-well polystyrene plates

85

(Immulon, Dynatech Laboratories Inc., Chantilly, VA) overnight at 4 8C. After washing with PBS/0.5% Tween, plates were blocked with 1% Telostean gelatin-PBS (Sigma), to inhibit non-specific binding, before loading of appropriately diluted serum or nasal secretion samples and incubation for 2 h at 37 8C. To determine IgA titers in nasal secretions, the samples were pretreated with 10 mM 1–4 dithiothreitol (Sigma) at 37 8C for 1 h to reduce the mucin content. Each plate also included an EHV-1 negative control serum, and a serially diluted positive control serum. Following incubation with samples, plates were washed and incubated with anti-equine immunoglobulin isotype-specific monoclonal antibodies (antiIgGa, IgGb, IgG(T), or IgA). These monoclonal antibodies were characterized during the Second International Workshop on Equine Leukocyte Antigens (specific antibody workshop number in parentheses): IgGa (WS29), IgGb (WS13), IgG(T) (WS30), and IgA (WS15) (Lunn et al., 1998). Plates were washed again prior to incubation with peroxidase conjugated AffinipureTM goat anti-mouse IgG & IgM(H + L) (Jackson Immuno Research Laboratories Inc., West Grove, PA). Color was developed by addition of 3,30 ,5,50 -tetramethybenzidine substrate (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD) and measured spectrophotometrically at 450 nm using an ELISA reader (EE 311SX Microplate Autoreader, Bio Tek Instruments, Winooski, VT). Titers of the samples were determined by comparison to a standard curve generated by a sample of a known titer. In addition, antibodies specific for either EHV-1 or EHV-4 in sera of all ponies was measured using a gG specific ELISA as previously described (Crabb et al., 1995). This test was performed to detect any accidental EHV-1 infection or reactivation from latency, since gG glycoprotein was not included in either vaccine. Because most animals had pre-existing titers for EHV, we also performed ELISA’s that detected changes in anti-EHV gC and gD antibodies as previously described (Ruitenberg et al., 2000a). Briefly, gC or gD antigen was coated onto 96-well flat bottomed microtiter plates (Immulon, Dynatech Laboratories Inc.), and plates were blocked with 3% BSA in PBS for 1 h. Test serum samples were diluted 1:500 in PBS/Tween/0.5% bovine serum albumin (BSA) (Sigma) prior to incubation on plates. Subsequently, plates were incubated with horseradish

86

G. Soboll et al. / Veterinary Immunology and Immunopathology 111 (2006) 81–95

peroxidase-conjugated anti-equine IgG (Sigma) color was developed with O-phenylenediamine dihydrochloride (Sigma), and the reaction was stopped with 3 M HCL. Absorbances were read spectrophotometrically at A = 450 and expressed as the mean OD value of triplicate serum dilutions. 2.11. Lymphoproliferation following in vitro restimulation of PBMCs with EHV-1 For determination of EHV-1 specific memory responses, PBMCs were collected and cryopreserved prior to the initial vaccination, 14 days after each vaccination, and 10 and 21 days after challenge infection. For in vitro re-stimulation, PBMCs were plated in triplicate in 96-well microtiter plates (Costar, Cambridge, MA), at a concentration of 2
105 cells in 200 ml per well, and re-stimulated with 2
106 PFU of heat inactivated EHV-1/A183. Controls included PBMCs incubated with RPMI-10 without virus and PBMCs stimulated with 5 mg/ml of PHA (Sigma). Plates were incubated in a humidified 37 8C, 5% CO2 incubator for a total of 6 days, with 1 mCi 3Hthymidine/well being added for the last 10 h. Lymphoproliferation was determined by measuring the Thymidine uptake using a microplate scintillation and luminescence counter system (Top Count, Packard, Meriden, CT). Proliferation was expressed as a stimulation index (SI), which was calculated by dividing the mean thymidine incorporation of stimulated cells, by the mean thymidine incorporation of unstimulated cells. 2.12. Cytotoxic T-lymphocyte responses EHV-1 specific cytotoxic activity of effectors contained in PBMCs was determined prior any vaccination, 2 weeks after each vaccination and 10 and 21 days post-challenge infection. CTL precursors were stimulated with 107 PFU of EHV-1/A183 per 3– 5
107 PBMCs for 6 days at 37 8C, 4% CO2. Virus specific killing was measured using virus or mock infected, autologous PWM lymphoblasts in a standard 4-h chromium release assay as previously described (Allen et al., 1995; Soboll et al., 2003c). In order to show that the killing was ELA-A restricted, virus stimulated effectors from each pony were tested against heterologous target cells.

2.13. Statistical analysis For examination of the effect of vaccination and challenge infection on the different immune responses, paired T-test comparisons between Day 0 and post-vaccination/challenge data were performed. In addition, differences between the three groups postchallenge infection were analyzed by ANOVA. Antibody data was log transformed before analysis and CTL data was transformed using arcsine (square root of % specific lysis/100).

3. Results 3.1. EHV-1 latency Prior any treatment, eleven ponies tested positive in the second round of a gB specific nested PCR prior to immunization or experimental challenge infection. Four ponies tested negative and these were from the two vaccination groups (ponies #45, 47, 54, and 59). 3.2. MHC class I typing The results of the MHC class I typing are shown in Table 1. The reagents for this test are polyclonal antisera, therefore there is likely subtypic variation within each serotype that reflects greater genetic complexity among horses. Furthermore, the available antisera did not cover all known alleles, thus horses that possess only one type may be homozygous or heterozygous for an undetected allele. Similarly, some ponies, such as Pony 55, show very little reactivity with any of the available antisera. 3.3. Clinical signs following EHV-1 challenge infection All ponies showed some signs of disease following challenge infection. Most ponies were febrile on Day 1 after challenge infection, with fevers lasting between 1 and 3 days (Fig. 1). A second fever spike was seen in most ponies for 1 day only, between Days 5 and 8, after challenge infection. Nasal and ocular discharge in the individual ponies following challenge infection is shown in Table 2. The mean duration of ocular and nasal discharge after challenge infection was 7 days

G. Soboll et al. / Veterinary Immunology and Immunopathology 111 (2006) 81–95

87

Table 1 MHC I typing (x5 or x11 indicate that there was reactivity with only some of the A5 or W11 antisera, respectively) Controls

Pony 10

Pony 17

Pony 95

Pony 99

Pony 58

ELA-A

A3/W11

A4/A5

A4/A5

A9/W11

A4/A5

gB/gC/gD DNA vacc. ELA-A

Pony 45 A1/A5

Pony 51 A4/W11

Pony 55 x11

Pony 57 A5/W11

Pony 59 A6

IE/UL5 DNA vacc. ELA-A

Pony 46 A6/x5

Pony 47 A4/A6

Pony 52 A3/A6

Pony 53 A3/A5

Pony 54 A5

Fig. 1. Rectal temperature on days post-challenge infection in 8F. Data are shown for individual ponies in different experimental groups.

88

G. Soboll et al. / Veterinary Immunology and Immunopathology 111 (2006) 81–95

Table 2 Ocular and nasal discharge following challenge infection: (–) none, (1) mild serous, (2) mucoid, (3) mucopurolent, and (4) copious mucopurolent Days postinfection

Controls 10

17

95

99

58

gB/gC/gD DNA vaccinates 45

51

55

57

59

IE/UL5 DNA vaccinates 46

47

52

53

54

0 1 2 3 4 5 6 7 8 9 1 12 14 16 18 21

– – – 3 4 4 4 4 3 – – 3 4 4 3 –

– 2 1 – 1 1 – 1 – – – 1 1 – 1 –

– 2 – 1 – – – – – 2 – – – – – –

– 1 2 – 2 – 1 1 – 1 – – – – – –

– – – 1 – 1 – 1 2 – – 2 1 1 1 –

– 1 1 – 1 2 2 1 – – – – – – – –

– – – – – 1 – – – – 1 – 1 – 1 1

– – – 2 4 4 4 4 4 4 4 3 4 3 – –

– – 2 2 1 1 2 1 2 3 1 – – 1 1 1

– – – – – – – – – – – – – – – 1

– 1 1 – – – – – – – – – – – 1 –

– – – – 1 – 2 – 1 – – – 3 – – –

– 2 2 – 2 1 3 – – – 1 – – – – –

– – – – 1 – – – – – – – – – – –

– – – – – – 1 – – – – – – – – –

total in the controls and in the gB/gC/gD DNA vaccinates. In contrast, mean duration of discharge for the IE/UL5 DNA group was only 3 days total (ranging from 1 to 6 days). Ponies in all groups were slightly depressed on the days they had high fevers, but maintained their appetites. No spontaneous coughing was observed in any pony. 3.4. Virus shedding and viremia The results of nasal virus isolation after challenge infection are shown in Fig. 2. No virus was isolated beyond 5 days post-infection in any pony. In control and vaccinatged ponies, virus shedding was seen in four of five ponies for durations of 1–4 days. A sample was counted positive for viremia when cytopathic effect was clearly evident in the second passage in cell culture. Virus could be detected in blood lymphocytes in one control pony on Day 16 following challenge infection. In the DNA vaccination groups, viremia was detected in four of five ponies in each group between Days 3 and 8 post-infection. The duration of viremia ranged from 1 to 3 days. 3.5. Antibody responses No increases in EHV-1 gG-specific antibody were detected in any group prior to challenge infection,

Fig. 2. EHV-1 shedding on days post-challenge infection. Samples were collected into viral transport media from the nasopharynx using Dacron swabs. Results are expressed as log PFU/ml of transport media. Each bar represents an individual pony; no virus shedding was detected beyond Day 5 post-challenge infection.

G. Soboll et al. / Veterinary Immunology and Immunopathology 111 (2006) 81–95

indicating that no accidental exposure with EHV-1 or reactivation of latent EHV-1 infection occurred. Following challenge infection, EHV-1 antibody titers to glycoprotein G titers rose significantly in all three groups (data not shown). In contrast, EHV-4 gG levels were consistently elevated in all ponies throughout the experiment and no fluctuations were seen. The isotype specific ELISA for detection of serum IgGa, IgGb, and IgG(T) and nasal IgA directed against EHV-1 virion proteins showed similar results

89

(Fig. 3). There were no significant increases above background levels for any isotype prior to challenge infection. Titers rose significantly in each group following challenge infection; however, differences between the three groups were not significant. Results for virus neutralizing antibodies also confirmed this trend. No significant responses were seen following vaccination, but titers increased significantly in all three groups following challenge infection (Fig. 3).

Fig. 3. Antibody isotype responses and virus neutralizing titers. The closed arrows represent time points of vaccination on Days 0, 41, 86, and 126. The open arrow represents EHV-1 challenge infection on Day 153. Results shown are means  S.E.M., n = 5 in each group.

90

G. Soboll et al. / Veterinary Immunology and Immunopathology 111 (2006) 81–95

In contrast, the gD specific ELISA showed that in the gB/gC/gD DNA vaccination group, three of five ponies responded with increased anti-gD antibody responses following vaccination (Fig. 4). These three ponies were those that had low titers prior to the start of the study, while the other two ponies in this group had high initial titers. None of the control ponies or ponies from the IE/UL5 DNA vaccination group responded with increases of gD specific antibody titers, indicating that the increases in the gB/gC/gD DNA vaccination group were specific to the vaccination and not a result of a recrudescing EHV-1 or EHV4 infection. The gC specific ELISA showed increases

Fig. 5. Mean EHV-1 specific lymphoproliferative responses. Results shown are mean SI  S.E.M., n = 5 in each group. Samples were collected prior to vaccination, 14 days post-third vaccination and 10 and 21 days post-challenge infection, respectively. Statistical significance between each time point and the time point 0 in the same experimental group is indicated as (*) for p < 0.05.

in the same three ponies of the gB/gC/gD DNA vaccination group as did the gD specific ELISA. However, responses were lower overall (data not shown). 3.6. Lymphoproliferative responses to viral restimulation EHV-1 specific lymphoproliferation at time points as indicated in the figure, are shown in Fig. 5. Neither vaccination group responded with significant increases in EHV-1 specific proliferation following vaccination. All three groups showed increases in EHV-1 specific proliferation following challenge infection; however, these increases were only significantly different from pre-vaccination levels in the IE/UL5 DNA vaccinates. 3.7. CTL responses

Fig. 4. Glycoprotein D specific antibody responses in individual ponies. Samples were collected prior to vaccination, and 14 days post-vaccinations or challenge infection, respectively.

There were no significant increases in EHV-1 specific CTL activity following vaccination in any group of ponies. Ponies of all three groups showed significant increases in EHV-1 specific cytotoxicity following challenge infection (Fig. 6). These increases were seen at effector: target ratios of 100:1, 33:1, and 11:1. All effectors were also tested against mock infected targets but cytotoxicity was not seen at any effector to target ratio in these assays. In addition effectors were tested against heterologous (ELA-class 1 mismatched) EHV-1 infected targets and no lysis was seen.

G. Soboll et al. / Veterinary Immunology and Immunopathology 111 (2006) 81–95

91

Fig. 6. EHV-1 specific CTL responses at different effector to target ratios. Results shown are means  S.E.M., n = 5 in each group. Samples were collected prior to vaccination, 14 days after each vaccination and 10 and 21 days post-challenge infection. Statistical significance between each time point and the time point 0 in the same experimental group is indicated as (*) for p < 0.05.

4. Discussion This is the first study to examine the value of particle-mediated DNA vaccination for protection from EHV-1 infection in ponies. There was only limited evidence that the DNA vaccination regime had an immunogenic effect in terms of antibody, proliferative, and CTL responses following vaccination. When using an ELISA system specific for the gD and gC glycoproteins we did detect increased antibody production in three of five ponies following vaccination with gB/gC/gD DNA. These three ponies had low initial gC/gD antibody titers. No effect of vaccination on the immune response was seen in ponies of the IE/

UL5 DNA vaccination group with any assay system following vaccination. Nevertheless, following challenge infection, ponies in this group showed a reduction in duration of nasal and ocular discharge from 7 to 3 days when compared with the control ponies, although there was no evidence of reduced pyrexia. This is consistent with results of a study in mice where intramuscular vaccination with IE DNA offered limited clinical protection (Koen et al., 2000). While previous studies in our laboratory have found particle-mediated DNA vaccination with the equine influenza virus hemagglutinin (HA) gene to be effective in stimulating antibody responses and protection in ponies, there are differences between

92

G. Soboll et al. / Veterinary Immunology and Immunopathology 111 (2006) 81–95

the use of this technology for equine influenza virus and EHV-1 vaccination. The influenza virus HA protein has long been recognized as an important vaccine component. In contrast no single gene with these properties has so far been identified for EHV-1. In addition, the HA gene is known to be immunogenic (Olsen et al., 1997), which might not be the case with the EHV-1 proteins. Another factor is that the vaccinated ponies were all latently infected with EHV-1, while ponies used in our influenza study were influenza naı¨ve. It is possible that more sensitive assay systems are needed in order to differentiate between immunity due to prior exposure and vaccine-induced immunity, although it might be anticipated that a protective immune response would in all likelihood still be detectable. Latent infection is common in the equine population (Edington et al., 1994), and this is a circumstance that any EHV-1 vaccination regime must overcome. There would be advantages to performing these experiments in EHV-1 specific pathogen free foals (Chong and Duffus, 1992), but the cost and difficulty in producing sufficient animals makes such experiments impractical. In contrast to our results, a study by our colleagues in Australia showed induction of gD-specific antibodies as well as VN antibodies and IgGa and IgGb antibodies following intramuscular gD DNA vaccination of horses with pre-existing antibodies (Ruitenberg et al., 2000a). However, the amount of gD DNA delivered in the Ruitenberg study (50, 200, and 500 mg), played a significant role in that the incidence of responses was highest in the group vaccinated with 500 mg of DNA. Even though particle-mediated DNA vaccination has been reported to be more efficient than intramuscular DNA vaccination (Fynan et al., 1995), the total amount of DNA per gene in our vaccination study was only 25 mg. Consistent with this idea was a study in mice showing dose dependence between the amounts of influenza virus nucleoprotein DNA given, and the specific CTL precursor and antibody responses elicited (Fu et al., 1999). In addition, a study of equine arteritis virus in horses that showed good seroconversion to a combination vaccination of gene gun and intramuscular injection used a total of 1.435 mg of DNA (Giese et al., 2002). This is almost 60 times more DNA than delivered in our experiment. While prior experience with particle-mediated DNA vaccination in horses would suggest that this would not be a barrier to

vaccination, the results of this study clearly indicate that for EHV-1 this is a factor. Although our DNA vaccination regime induced only limited immune responses, this study still gives a comprehensive overview of the events associated with EHV-1 equine respiratory infection in a group of young ponies. There are numerous studies that describe the individual immune effector mechanisms following EHV-1 infection in the horse (Allen et al., 1995; Breathnach et al., 2001; Gibson et al., 1992; Kydd et al., 1994a,b, 2003; O’Neill et al., 1999). However, this study provides an overview of how these immune effector mechanisms relate to each other in ponies with pre-existing EHV-1 immunity. In this group of 15 ponies, 11 were latently infected as determined by nested PCR. However, prior to initial vaccination, all 15 had some EHV-1 specific antibody levels as determined by a variety of tests. Fourteen of 15 showed some level of CTL responses, and 10 of 15 showed EHV-1 specific proliferative responses. No pony was consistently negative with all tests; in fact the data of the different tests did not seem to correlate with each other implying that there are sensitivity and/ or specificity issues with most assay systems and that one should not rely on a single test when examining pre-existing immunity or exposure to EHV-1. The prevalence of ponies with pre-existing immunity in our study is consistent with studies in the UK and Australia that reported 87.5% and 81.8% of horses to be latently infected with EHV-1 and EHV-4 (Edington et al., 1994; Gilkerson et al., 1999a) and that the establishment of latency most likely occurs within the first year of life (Gilkerson et al., 1999a). EHV-1 specific antibodies were measured by a variety techniques in this study. Antibody responses measured by all assays, except for the gC and gD specific ELISA’s, remained low until challenge infection. EHV-1 specific IgGa and IgGb were the predominant isotype responses in serum, and IgA was predominant in nasal secretions. This is consistent with studies by others (Breathnach et al., 2001; Ruitenberg et al., 2000a). The VN titers appeared to correlate well with IgGb isotype responses. Ponies in all groups responded to challenge infection with increased EHV-1 specific proliferative and CTL responses. No association between these immune responses and viremia, clinical data, or antibody titers could be found. However, it has been

G. Soboll et al. / Veterinary Immunology and Immunopathology 111 (2006) 81–95

reported previously that bulk CTL assays are not sensitive enough to predict protection. Limiting dilution assays (LDA) determining CD8+ cytotoxic T lymphocyte precursor frequencies have so far been found to be the only correlate of protection (Allen et al., 1995; Kydd et al., 2003; O’Neill et al., 1999). LDAs are however difficult to perform and time consuming and the development of IFN-g based assays might make the examination of cellular immune responses to EHV-1 easier. A correlation between MHC haplotype and immunity to EHV-1 has been reported (Bodo et al., 1994; Soboll et al., 2003a,b). More specifically we have shown that the IE proteins induces CTL responses in PBMCs of ponies carrying an A3.1 ELA-A allele. In this experiment, none of the ponies carried the A3.1 allele (all the A3 ponies expressed the A3.2 allele), which could explain why we did not see an induction in CTL responses following vaccination with the IE/UL5 vaccine. At the time this study was initiated the information with regard the A3.1/IE relationship was not available, and ponies were assigned to vaccination groups independent of their MHC I haplotype. The heterogeneity of the vaccination groups in regards to ELA haplotype may also have masked more positive results in individual ponies and MHC haplotype clearly needs to be a consideration for future vaccination trials. In summary, particle-mediated EHV-1 DNA vaccination at a dose of 25 mg of DNA/plasmid/ vaccination induced limited immune responses in ponies, and did not protect from challenge infection. Only after EHV-1 infection were significant antibody and cellular immune responses induced. Despite the success of particle-mediated DNA vaccination of ponies against another important equine viral respiratory infection, influenza virus, this strategy shows little promise for protection against EHV-1. Several conclusions can be drawn from these observations. Firstly, successful results of similar studies on DNA vaccination in mice do not necessarily predict success in horses. Secondly, choice of EHV-1 genes and the influence of MHC-I restriction of immune responses may limit the value of vaccination with limited sets of viral genes. Thirdly, even when available, particlemediated DNA vaccination of skin and mucosal surfaces is not universally successful in generating high titered antibody responses to viral glycoproteins.

93

Future vaccines aiming to prevent both primary respiratory infection and cell-associated viremia will likely have to induce cellular immune responses, and different strategies such as a prime/boost vaccination, recombinant vectors, or modified live vaccines may have a better chance of achieving this goal. Acknowledgments This study was supported by grants from the USDA and the Grayson-Jockey Club Research Foundation. References Allen, G.P., Kydd, J.H., Slater, J.D., Smith, K.C., 1999. Advances in understanding of the pathogenesis, epidemiology, and immunological control of equid herpesvirus abortion. In: Wernery, U., Wade, J.F., Mumford, J.A., Kaaden, O.-R. (Eds.), Equine Infectious Diseases VIII. Proceedings of the Eighth International Conference, Dubai 23–26 March, 1998. R & W Publications, Newmarket, pp. 129–146. Allen, G.P., Yeargan, M., Costa, L.R., Cross, R., 1995. Major histocompatibility complex class I-restricted cytotoxic T-lymphocyte responses in horses infected with equine herpesvirus 1. J. Virol. 69, 606–612. Allen, G.P., Yeargan, M.R., 1987. Use of lambda gt11 and monoclonal antibodies to map the genes for the six major glycoproteins of equine herpesvirus 1. J. Virol. 61, 2454–2461. Antczak, D.F., 1992. The major histocompatibility complex of the horse. In: Plowright, W., Rossdale, P.D., Wade, J.F. (Eds.), Equine Infectious Diseases IV. Proceedings of the Sixth International Conference, Cambridge 1991. R & W Publications, Newmarket, pp. 99–112. Audibert, F.M., Lise, L.D., 1993. Adjuvants: current status, clinical perspectives and future prospects. Trends Pharmacol. Sci. 14, 174–178. Bailey, E., Antczak, D.F., Bernoco, D., Bull, R.W., Fister, R., Guerin, G., Lazary, S., Matthews, S., McClure, J., Meyer, J., 1984.In: Joint Report of the Second International Workshop on Lymphocyte Alloantigens of the Horse, 3–8 October 1982, vol. 15, Animal Blood Groups & Biochemical Genetics, pp. 123–132. Bailey, E., Marti, E., Fraser, D.G., Antczak, D.F., Lazary, S., 2000. Immunogenetics of the horse. In: Bowling, A.T., Ruvinsky, A. (Eds.), The Genetics of the Horse. CABI Publishing, New York, pp. 123–155. Banchereau, J., Steinman, R.M., 1998. Dendritic cells and the control of immunity. Nature 392, 245–252. Bhardwaj, N., Bender, A., Gonzalez, N., Bui, L.K., Garrett, M.C., Steinman, R.M., 1995. Stimulation of human anti-viral CD8+ cytolytic T lymphocytes by dendritic cells. Adv. Exp. Med. Biol. 378, 375–379 (review). Bodo, G., Marti, E., Gaillard, C., Weiss, M., Bruckner, L., Gerber, H., Lazary, S., 1994. Association of the immune response with

94

G. Soboll et al. / Veterinary Immunology and Immunopathology 111 (2006) 81–95

the major histocompatibility complex of the horse. In: Nakajima, H., Plowright, W. (Eds.), In Equine Infectious Diseases VII. R & W Publications, Newmarket, pp. 143–152. Borchers, K., Slater, J., 1993. A nested PCR for the detection and differentiation of EHV-1 and EHV-4. J. Virol. Methods 45, 331– 336. Breathnach, C.C., Yeargan, M.R., Sheoran, A.S., Allen, G.P., 2001. The mucosal humoral immune response of the horse to infective challenge and vaccination with equine herpesvirus-1 antigens. Equine Vet. J. 33, 651–657. Bryans, J.T., Allen, G.P., 1989. Herpesviral diseases of the horse. In: Wittmann, G. (Ed.), Herpesvirus Diseases of Cattle, Horses, and Pigs. Kluwer Academic Publishers, Boston, pp. 176–229. Burki, F., Rossmanith, W., Nowotny, N., Pallan, C., Mostl, K., Lussy, H., 1990. Viraemia and abortions are not prevented by two commercial equine herpesvirus-1 vaccines after experimental challenge of horses. Vet. Q. 12, 80–86. Chong, Y.C., Duffus, W.P., 1992. Immune responses of specific pathogen free foals to EHV-1 infection. Vet. Microbiol. 32, 215– 228. Cohen, J., 1993. Naked DNA points way to vaccines. Science 259, 1691–1692. Crabb, B.S., Allen, G.P., Studdert, M.J., 1991. Characterization of the major glycoproteins of equine herpesviruses 4 and 1 and asinine herpesvirus 3 using monoclonal antibodies. J. Gen. Virol. 72, 2075–2082. Crabb, B.S., MacPherson, C.M., Reubel, G.H., Browning, G.F., Studdert, M.J., Drummer, H.E., 1995. A type-specific serological test to distinguish antibodies to equine herpesviruses 4 and 1. Arch. Virol. 140, 245–258. Doll, E.R., Crowe, M.E.W., Bryans, J.T., McCollum, W.H., 1955. Infection immunity in equine virus abortion. Cornell Vet. 45, 387–410. Edington, N., Welch, H.M., Griffiths, L., 1994. The prevalence of latent Equid herpesviruses in the tissues of 40 abattoir horses. Equine Vet. J. 26, 140–142. Ellis, J.A., Bogdan, J.R., Kanara, E.W., Morley, P.S., Haines, D.M., 1995. Cellular and antibody responses to equine herpesviruses 1 and 4 following vaccination of horses with modified-live and inactivated viruses. J. Am. Vet. Med. Assoc. 206, 823–832 (see comments). Flowers, C.C., Flowers, S.P., Sheng, Y., Tarbet, E.B., Jennings, S.R., O’Callaghan, D.J., 1995. Expression of membrane-bound and secreted forms of equine herpesvirus 1 glycoprotein D by recombinant baculovirus. Virus Res. 35, 17–34. Fu, T.M., Guan, L., Friedman, A., Schofield, T.L., Ulmer, J.B., Liu, M.A., Donnelly, J.J., 1999. Dose dependence of CTL precursor frequency induced by a DNA vaccine and correlation with protective immunity against influenza virus challenge. J. Immunol. 162, 4163–4170. Fynan, E.F., Webster, R.G., Fuller, D.H., Haynes, J.R., Santoro, J.C., Robinson, H.L., 1995. DNA vaccines: a novel approach to immunization. Int. J. Immunopharmacol. 17, 79–83 (review). Gallichan, W.S., Rosenthal, K.L., 1996. Long-lived cytotoxic T lymphocyte memory in mucosal tissues after mucosal but not systemic immunization. J. Exp. Med. 184, 1879–1890.

Gibson, J.S., Slater, J.D., Awan, A.R., Field, H.J., 1992. Pathogenesis of equine herpesvirus-1 in specific pathogen-free foals: primary and secondary infections and reactivation. Arch. Virol. 123, 351–366. Giese, M., Bahr, U., Jakob, N., Kehm, R., Handermann, M., Mueller, S.N., Vahlenkamp, T., Spies, T., Schneider, T., Schusser, G., Darai, G., 2002. Stable and long-lasting immune responses in horses after DNA vaccination against equine arteritis virus. Virus Genes 25, 159–167. Gilkerson, J.R., Whalley, J.M., Drummer, H.E., Studdert, M.J., Love, D.N., 1999a. Epidemiological studies of equine herpesvirus 1 (EHV-1) in thoroughbred foals: a review of studies conducted in the Hunter Valley of New South Wales between 1995 and 1997. Vet. Microbiol. 68, 15–25. Gilkerson, J.R., Whalley, J.M., Drummer, H.E., Studdert, M.J., Love, D.N., 1999b. Epidemiology of EHV-1 and EHV-4 in the mare and foal populations on a Hunter Valley stud farm: are mares the source of EHV-1 for unweaned foals. Vet. Microbiol. 68, 27–34. Hassett, D.E., Whitton, J.L., 1996. DNA immunization. Trends Microbiol. 8, 307–312. Koen, M.T., Walker, C., Wellington, J.E., Love, D.N., Whalley, J.M., 2000. Characterisation of IE and UL5 gene products of equine herpesvirus 1 using DNA inoculation of mice. Arch. Virol. 145, 2677–2686. Kukreja, A., Love, D.N., Whalley, J.M., Field, H.J., 1998. Study of the protective immunity of co-expressed glycoprotein H and L of equine herpesvirus-1 in a murine intranasal infection model. Vet. Microbiol. 60, 1–11. Kydd, J.H., Smith, K.C., Hannant, D., Livesay, G.J., Mumford, J.A., 1994a. Distribution of equid herpesvirus-1 (EHV-1) in respiratory tract associated lymphoid tissue: implications for cellular immunity. Equine Vet. J. 26, 470–473. Kydd, J.H., Smith, K.C., Hannant, D., Livesay, G.J., Mumford, J.A., 1994b. Distribution of equid herpesvirus-1 (EHV-1) in the respiratory tract of ponies: implications for vaccination strategies. Equine Vet. J. 26, 466–469 (see comments). Kydd, J.H., Wattrang, E., Hannant, D., 2003. Pre-infection frequencies of equine herpesvirus-1 specific, cytotoxic T lymphocytes correlate with protection against abortion following experimental infection of pregnant mares. Vet. Immunol. Immunopathol. 96, 207–217. Ludewig, B., Ehl, S., Karrer, U., Odermatt, B., Hengartner, H., Zinkernagel, R.M., 1998. Dendritic cells efficiently induce protective antiviral immunity. J. Virol. 72, 3812–3818. Lunn, D.P., Holmes, M.A., Antczak, D.F., Baker, J.M., BendaliAhcene, S., Blanchard-Channell, M., Byrne, K.M., Cannizzo, K., Davis, W., Hamilton, M.J., Hannant, D., Kondo, T., Kydd, J.H., Monier, M.C., Moore, P.F., Neeraj, N., Schram, B.R., Sheoran, A.S., Stott, J.L., Sugiura, T., Vagnoni, K.E., 1998. Report of the Second Equine Leucocyte Antigen Workshop. Vet. Immunol. Immunopathol. 62, 101–143. Lunn, D.P., Soboll, G., Schram, B.R., Quass, J., McGregor, M.W., Drape, R., Macklin, M.D., McCabe, D.E., Swain, W.F., Olsen, C.W., 1999. Antibody responses to DNA vaccination of horses using the influenza virus hemagglutinin gene. Vaccine 17, 2245– 2258.

G. Soboll et al. / Veterinary Immunology and Immunopathology 111 (2006) 81–95 Mumford, J.A., Rossdale, P.D., Jessett, D.M., Gann, S.J., Ousey, J., Cook, R.F., 1987. Serological and virological investigations of an equid herpesvirus 1 (EHV-1) abortion storm on a stud farm in 1985. J. Reprod. Fertil. Suppl. 35, 509–518. Munro, K.I., Wellington, J.E., Love, D.N., Whalley, J.M., 1999. Characteristics of glycoprotein B of equine herpesvirus 1 expressed by a recombinant baculovirus. Vet. Microbiol. 68, 49–57. O’Neill, T., Kydd, J.H., Allen, G.P., Wattrang, E., Mumford, J.A., Hannant, D., 1999. Determination of equid herpesvirus 1-specific, D8+ cytotoxic T lymphocyte precursor frequencies in ponies. Vet. Immunol. Immunopathol. 70, 43–54. Oehen, S., Hengartner, H., Zinkernagel, R.M., 1991. Vaccination for disease. Science 251, 195–198. Olsen, C.W., McGregor, M.W., Dybdahl-Sissoko, N., Schram, B.R., Nelson, K.M., Lunn, D.P., Macklin, M.D., Swain, W.F., Hinshaw, V.S., 1997. Immunogenicity and efficacy of baculovirusexpressed and DNA-based equine influenza virus hemagglutinin vaccines in mice. Vaccine 15, 1149–1156. Osterrieder, N., Wagner, R., Brandmuller, C., Schmidt, P., Wolf, H., Kaaden, O.R., 1995. Protection against EHV-1 challenge infection in the murine model after vaccination with various formulations of recombinant glycoprotein gp14 (gB). Virology 208, 500–510. Packiarajah, P., Walker, C., Gilkerson, J., Whalley, J.M., Love, D.N., 1998. Immune responses and protective efficacy of recombinant baculovirus-expressed glycoproteins of equine herpesvirus 1 (EHV-1) gB, gC and gD alone or in combinations in BALB/c mice. Vet. Microbiol. 61, 261–278. Rouse, R.J., Nair, S.K., Lydy, S.L., Bowen, J.C., Rouse, B.T., 1994. Induction in vitro of primary cytotoxic T-lymphocyte responses with DNA encoding herpes simplex virus proteins. J. Virol. 68, 5685–5689. Ruitenberg, K.M., Love, D.N., Gilkerson, J.R., Wellington, J.E., Whalley, J.M., 2000a. Equine herpesvirus 1 (EHV-1) glycoprotein D DNA inoculation in horses with pre-existing EHV-1/ EHV-4 antibody. Vet. Microbiol. 76, 117–127. Ruitenberg, K.M., Walker, C., Love, D.N., Wellington, J.E., Whalley, J.M., 2000b. A prime-boost immunization strategy with DNA and recombinant baculovirus-expressed protein enhances protective immunogenicity of glycoprotein D of equine herpesvirus 1 in naïve and infection-primed mice. Vaccine 18, 1367–1373. Ruitenberg, K.M., Walker, C., Wellington, J.E., Love, D.N., Whalley, J.M., 1999a. DNA-mediated immunization with glycoprotein D of equine herpesvirus 1 (EHV-1) in a murine model of EHV-1 respiratory infection. Vaccine 17, 237–244. Ruitenberg, K.M., Walker, C., Wellington, J.E., Love, D.N., Whalley, J.M., 1999b. Potential of DNA-mediated vaccination for equine herpesvirus 1. Vet. Microbiol. 68, 35–48.

95

Siedek, E.M., Whelan, M., Edington, N., Hamblin, A., 1999. Equine herpesvirus type 1 infects dendritic cells in vitro: stimulation of T lymphocyte proliferation and cytotoxicity by infected dendritic cells. Vet. Immunol. Immunopathol. 67, 17–32. Smith, P.M., Zhang, Y., Jennings, S.R., O’Callaghan, D.J., 1998. Characterization of the cytolytic T-lymphocyte response to a candidate vaccine strain of equine herpesvirus 1 in CBA mice. J. Virol. 72, 5366–5372. Soboll, G., Horohov, D.W., Aldridge, B.M., Olsen, C.W., McGregor, M.W., Drape, R.J., Macklin, M.D., Swain, W.F., Lunn, D.P., 2003a. Regional antibody and cellular immune responses to equine influenza virus infection, and particle mediated DNA vaccination. Vet. Immunol. Immunopathol. 94, 47–62. Soboll, G., Nelson, K.M., Leuthner, E.S., Clark, R.J., Drape, R., Macklin, M.D., Swain, W.F., Olsen, C.W., Lunn, D.P., 2003b. Mucosal co-administration of cholera toxin and influenza virus hemagglutinin-DNA in ponies generates a local IgA response. Vaccine 21, 3081–3092. Soboll, G., Whalley, J.M., Koen, M.T., Allen, G.P., Fraser, D.G., Macklin, M.D., Swain, W.F., Lunn, D.P., 2003c. Identification of equine herpesvirus-1 antigens recognized by cytotoxic T lymphocytes. J. Gen. Virol. 84, 2625–2634. Stokes, A., Alber, D.G., Cameron, R.S., Marshall, R.N., Allen, G.P., Killington, R.A., 1996. The production of a truncated form of baculovirus expressed EHV-1 glycoprotein C and its role in protection of C3H (H-2Kk) mice against virus challenge. Virus Res. 44, 97–109. Tewari, D., Whalley, J.M., Love, D.N., Field, H.J., 1994. Characterization of immune responses to baculovirus-expressed equine herpesvirus type 1 glycoproteins D and H in a murine model. J. Gen. Virol. 75, 1735–1741. van Der Meulen, K.M., Nauwynck, H.J., Buddaert, W., Pensaert, M.B., 2000. Replication of equine herpesvirus type 1 in freshly isolated equine peripheral blood mononuclear cells and changes in susceptibility following mitogen stimulation. J. Gen. Virol. 81, 21–25. Walker, C., Ruitenberg, K., Wellington, J., Whalley, J.M., Love, D.N., 1997. Genetic immunization with equine herpesvirus-1 glycoprotein D. First International Veterinary Vaccines and Diagnostics Conference, Madison, Wisconsin, July 1997, p. 93. Wellington, J.E., Lawrence, G.L., Love, D.N., Whalley, J.M., 1996a. Expression and characterization of equine herpesvirus 1 glycoprotein D in mammalian cell lines. Arch. Virol. 141, 1785–1793. Wellington, J.E., Love, D.N., Whalley, J.M., 1996b. Evidence for involvement of equine herpesvirus 1 glycoprotein B in cell–cell fusion. Arch. Virol. 141, 167–175. Whalley, J.M., Love, D.N., Tewari, D., Field, H.J., 1995. Characteristics of equine herpesvirus 1 glycoproteins expressed in insect cells. Vet. Microbiol. 46, 193–201.