Prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant Moraxella bovis cytotoxin-ISCOM matrix adjuvanted vaccine

Vaccine 23 (2004) 537–545

Prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant Moraxella bovis cytotoxin-ISCOM matrix adjuvanted vaccine John A. Angelosa,∗ , John F. Hessb , Lisle W. Georgea a

Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA b Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis, CA 95616, USA Received 8 December 2003; received in revised form 2 June 2004; accepted 4 June 2004 Available online 25 July 2004

Abstract The efficacy of a recombinant Moraxella bovis cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK) was evaluated in a randomized, blinded, controlled field trial. Ninety-three cross bred beef calves were vaccinated with either saline, ISCOM matrix (adjuvant control), or a recombinant M. bovis cytotoxin carboxy terminus peptide plus ISCOM matrix and boostered 21 days later. Ocular examinations were performed once weekly for 20 weeks. At week 12, the cumulative proportion of calves with ulcerated eyes in the recombinant vaccine group was significantly lower than in the saline control group. Throughout the 20 week trial, the cumulative proportion of ulcerated calves remained lowest in the recombinant vaccine group. By week 7, nonulcerated calves in the recombinant vaccine group had significantly higher changes in serum neutralizing titers and cytotoxin specific to total IgG ratios in serum and tears as compared to calves in the control groups. The trend for a reduced cumulative proportion of IBK in the vaccinated calves over the 20 week trial suggests that a recombinant M. bovis cytotoxin vaccine may be beneficial in helping to prevent naturally occurring IBK. © 2004 Elsevier Ltd. All rights reserved. Keywords: Moraxella bovis; Cytotoxin; ISCOM

1. Introduction Infectious bovine keratoconjunctivitis (IBK; pinkeye) caused by Moraxella bovis [1] is the most common ocular disease of cattle. Cattle with IBK exhibit corneal ulceration, corneal edema, ocular pain, photophobia, and lacrimation. While most animals recover with varying degrees of corneal scarring, corneal rupture and permanent blindness may result in more severe cases. In addition to infection with M. bovis, environmental factors such as mechanical trauma to the cornea from seed awns, flies [2–4], and solar irradiation [5,6] are also linked to IBK. Both hemolytic and nonhemolytic strains of M. bovis exist in nature [7,8]. Pathogenicity has been associated with the expression of pilin proteins and a ∗

Corresponding author. Tel.: + 1 530 752 7407; fax: +1 530 752 0414. E-mail address: [email protected] (J.A. Angelos).

0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2004.06.025

hemolytic, cytolytic toxin (cytotoxin). The M. bovis cytotoxin is a RTX (repeats in the structural toxin) toxin [9] that is lytic for bovine neutrophils, erythrocytes, lymphocytes, corneal epithelial cells and causes corneal ulceration [8,10–13]. Previous investigators have examined pilin or cytotoxin as vaccine antigens to prevent IBK. Pilin proteins mediate attachment of M. bovis to the corneal surface [14–16] and pilus-based vaccines were effective at reducing incidence and severity of IBK [17–21]. The presence of at least seven different pilus serogroups [22] as well as the potential for pilin gene inversions [19,23], however, increases opportunities for antigenic variability and most likely accounts for the observed lack of pilus vaccine protection against challenge from heterologous M. bovis pilus serogroups [21]. Unlike pilins, the M. bovis cytotoxin appears to be more conserved amongst different strains. Hemolysin neutralizing antibodies develop in cattle with IBK [11,24–26] and can

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neutralize the hemolysin from diverse M. bovis strains [26]. Vaccination with partially purified M. bovis hemolysin also protected calves from challenge with heterologous isolates of M. bovis [24]. More recently, hemolytic and cytolytic activity of native M. bovis cytotoxin was shown to be neutralized using rabbit antisera raised against the carboxy terminal 284 amino acids of the M. bovis cytotoxin [9]. The neutralizing capacity of these antibodies suggested that a peptide vaccine might prevent IBK; the following study was done to examine the efficacy of a recombinant M. bovis cytotoxin vaccine to prevent naturally occurring IBK. 2. Materials and methods 2.1. Study population Animals for this study included 93 Angus or AngusHereford crossbred calves at the University of California Sierra Foothills Field Station, Brown’s Valley, CA. The criteria for selection of this farm included a high annual prevalence of IBK, adequate cattle handling facilities, and guarantees of cooperation from herd managers. Calves were pastured in fields containing either grass or mixed grasses and were moved between pastures as needed to maintain adequate nutritional intake. The study population was comprised of 20 bull calves, 71 steers, and 2 heifer calves ranging between 61 and 236 days of age and 97.2–292.7 kg. Prior to starting the trial, calves were dewormed, supplemented with selenium, and vaccinated against clostridial diseases, vibriosis, and leptospirosis. 2.2. Pre-enrollment examination Heads of calves were restrained and both eyes were examined. Calves with normally appearing eyes with corneas that were free of ocular scarring or opacification were randomly assigned to groups by use of a blocked randomization scheme. On day 0, calves that were chosen for enrollment were given 2 ml of either saline (0.9% sodium chloride injection USP, Baxter Healthcare Corp, Union, NJ), ISCOM matrix (adjuvant group), or a recombinant M. bovis cytotoxin carboxy terminus plus ISCOM matrix adjuvanted vaccine (recombinant cytotoxin group). The calves were boostered 21 days later. Study personnel were unaware of the contents of the vaccines that were administered. 2.3. Post-enrollment examination Calves were examined once a week for 20 weeks following the primary vaccination. Any eyes with corneal opacities were stained with fluorescein (Fluor-I-Strip, Ayerst Laboratories Inc., Philadelphia, PA) to determine if a corneal ulcer was present. Ulcers received a score of 0, 1, 2, or 3 based on the widest ulcer diameter. The scoring criteria were: 0 = no ulcer; 1 = an ulcer with the widest diameter <5 mm; 2 = an ulcer with the widest diameter ≥5 mm; and 3 = a perfo-

rated corneal ulcer. Ulcerated eyes were photographed with a ruler held next to the eye for measurement of the corneal ulcer surface area as described below. Calves were treated with florfenicol (40 mg/kg subcutaneously; Nuflor, ScheringPlough Animal Health, Union, NJ) if the ulcer score reached 2 or 3. To prevent iatrogenic infections, examiners wore rubber gloves, and plastic obstetrical sleeves/aprons that were rinsed with 1% chlorhexidine solution between calves. 2.4. Bacterial strains and genomic DNA M. bovis strain Tifton I was originally isolated from a beef cow with IBK in southern Georgia (L.W. George, unpublished). To prepare genomic DNA, M. bovis strain Tifton I was propagated in Luria–Bertani (LB) broth containing 1.5 mM calcium chloride with shaking at 37 ◦ C. Cells were pelleted, resuspended in 1/10th of the starting culture volume in a buffer containing 50 mM glucose, 25 mM Tris, 10 mM EDTA, pH 8, with 10 mg/ml lysozyme (Sigma, St. Louis, MO), and incubated at 37 ◦ C for 30 min. Sodium dodecyl sulfate (Sigma) was added to 0.6%, RNAse A was added to 10 ␮g/ml, Proteinase K was added to 1 mg/ml, and the solution was incubated for 1 h at 50 ◦ C. Protein was removed by phenol/chloroform extractions and genomic DNA was precipitated with NaCl/ethanol. Cloning competent Escherichia coli strains TOP10 (Invitrogen Corp., Carlsbad, CA), DH5␣ (Life Technologies Inc., Rockville, MD), and BL21 (DE3) (Novagen, Madison, WI) were propagated in LB broth or on LB agar (1.5% agar; Difco Laboratories, Detroit, MI). Antibiotic selection of E. coli was made using ampicillin (100 ␮g/ml) and kanamycin (50 ␮g/ml). 2.5. Expression of recombinant M. bovis cytotoxin Primers SNP down (5 -AAT GAC GAT ATC TTT GTT GGT CAA GGT AAA-3 ) and LNP2 (5 -TAG TAA ATT AAA TNA CTW AAC ACT-3 ) were used to amplify genomic DNA from M. bovis strain Tifton I. Amplifications were performed with Taq polymerase (Life Technologies Inc., Rockville, MD) using 30 cycles of 30 seconds each at 95 ◦ C, 1 min at 55 ◦ C, and 45 s at 72 ◦ C, followed by 10 min incubation at 72 ◦ C. The PCR product was cloned into pCR2.1-TOPO (TOPO TA cloning kit; Invitrogen Corp., Carlsbad, CA) and the resulting recombinant plasmid was digested with EcoRI. The appropriate fragment was gel purified (QIAquick Gel Extraction Kit, Qiagen) and cloned into bluntended, SmaI digested pT7-7 [27] to yield an expression construct that would direct synthesis of the cytotoxin gene from amino acids 590 through 927. Recombinant plasmids were transformed into E. coli DH5␣, and were subsequently purified prior to transformation into E. coli strain BL21 (DE3) for expression. Cells were grown to an OD600 of 0.8, and expression was induced by adding isopropylthio-␤-d-galactoside (IPTG; Life Technologies Inc.) to 1 mM. Induction was continued 9 h and cells were harvested by centrifugation. The

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expressed proteins formed inclusion bodies that were purified as described [28]. Purified inclusion bodies were solubilized in buffer containing 4 M urea, 0.25% Triton X-100, 5 mM Tris–HCl (pH, 7.5) and 1 mM EDTA and chromatographed (Mono Q column; Amersham Pharmacia Biotech Inc., Piscataway, NJ). Peak fractions were identified by SDS-PAGE, pooled, and protein was quantitated (BCA Kit; Pierce, Rockford, IL). For use as coating antigen in enzyme linked immunosorbent assays (ELISA), the affinity purified protein was stored frozen at −20 ◦ C. For vaccine use, the protein was filter sterilized and dialyzed against phosphate buffered saline (PBS) to remove urea, and stored at −20 ◦ C. During dialysis, the purified protein spontaneously precipitated. The protein concentration of the final product was based on the protein concentration in the sample before dialysis and was adjusted with PBS to deliver 500 ␮g per ml. 2.6. ISCOM matrix Immune stimulating complexes (ISCOMs) were prepared according to standard protocols [29] except that the recombinant protein was not incorporated directly into ISCOM particles, but rather was mixed with ISCOM matrices following matrix formation. Two ml of a lipid mixture containing 50 mg/ml each of L-␣-phosphatidylcholine (Sigma Chemical Co., St. Louis, MO) and 5-cholesten-3␤-ol (cholesterol, Sigma) in chloroform was added to a detergent solution made by dissolving 2 g decanoyl-N-methylglucamide (MEGA-10, ultrol grade, Calbiochem, La Jolla, CA) in 10 ml distilled water. This solution was added to PBS (pH 7.4; 0.1 ml lipid mix per 1 ml PBS) and the chloroform was removed by heating the solution to 45 ◦ C under vacuum until the solution cleared. Quil-A saponin (Superfos Biosector, Denmark) was added to a final concentration of 1 mg/ml. The resulting solution was dialyzed (2000 MWCO; Spectra/Por CE, Spectrum Medical Industries Inc., Houston, TX) for 3 days in 50 mM Tris–HCl, 0.001% thimerosal (Sigma), pH 7.5 at room temperature. Thimerosal was removed by dialysis against PBS (pH 7.4) for 24–36 h at 4 ◦ C. Following dialysis the solution was filter sterilized (0.45 or 0.2 ␮M pore size) and stored at −20 ◦ C until use. The presence of ISCOM matrices was confirmed by electron microscopic examination of negatively stained dialysate (Department of Medical Pathology, School of Medicine, UC Davis). The final 145 ml volume of ISCOM matrix was further diluted with PBS (pH 7.4) to 180 ml and stored at −80 ◦ C. 2.7. Vaccine formulation The final ISCOM matrix solution was mixed 1:1 with recombinant protein such that the final 2 ml vaccine dose delivered 500 ␮g recombinant protein. The ISCOM matrix solution mixed 1:1 with PBS as a 2 ml dose served as the adjuvant control. The saline control vaccine consisted of a 2 ml saline injection. Injections were administered subcutaneously in the neck.

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2.8. Corneal ulcer surface area measurement Color slides of each ulcer were projected to a standard magnification, the fluorescein stained areas were traced, the tracings were digitized, and ulcer areas were determined with public domain software (NIH Image program; available at http://rsb.info.nih.gov/nih-image). The average area determined from three tracings of each ulcer was used to represent the final corneal ulcer surface area. The limit of detection was 0.008 cm2 , an area corresponding to a 1 mm diameter circle. Ulcers with surface areas measuring <0.008 cm2 and those from mechanical trauma (corneal scratches or plant awns) were not included in the analyses. For data analysis, a square root transformation of the original ulcer surface area was used to represent the ulcer surface area measurement (SAM). The SAMs of ulcers that were induced by seed awns were only included in the analysis if the ulcer persisted through the time of a subsequent weekly examination. 2.9. Tear and serum collection Ocular secretions were collected prevaccination (week 0) and at week 7 (4 weeks post-booster). Tears from both eyes were collected and pooled; in ulcerated calves, tears were only collected from the nonulcerated eye. To collect tears, a cotton dental roll (Patterson Dental Supply, Inc, St. Paul, MN) was placed under the upper eyelid until the roll was saturated. Rolls were removed and placed over a solid support (1 cc syringe plunger) contained within a 15 ml transport tube (15 ml polypropylene Falcon tube, Becton Dickinson Labware, Franklin Lakes, NJ) and stored on ice. Fluid from the rolls was extracted by centrifugation, filter sterilized (Supor Acrodisc Syringe Filter (0.8/0.2 um); Pall Gelman Laboratory, Ann Arbor, MI), and stored frozen at −80 ◦ C until use. Tear total protein was quantitated with a commercial kit (BCA Protein Assay Reagent; Pierce, Rockford, IL). Whole blood was collected by jugular venipuncture into serum separator tubes (Corvac tubes, Sherwood-Davis & Geck, St. Louis, MO) and was stored on ice until the serum was harvested by centrifugation; serum was stored at −80 ◦ C until use. Prior to use in neutralization assays and ELISAs, serum and tear samples were heat inactivated at 56 ◦ C for 1 h. 2.10. Tear and serum hemolysis neutralization assays Cytotoxin was prepared from M. bovis strain Tifton I as previously described [9]; prior to use in neutralization assays, the cytotoxin was diluted 1:128 in chilled (4 ◦ C) Tris buffered saline-calcium chloride buffer (TBS CaCl2 buffer; 50 mM Tris, 150 mM NaCl, 1.5 mM CaCl2 (pH 7.4)). For neutralization assays, doubling dilutions of tear samples in TBS CaCl2 buffer were made in 96 well tissue culture plates (Nunc TC Microwell 96F, Nalge Nunc International, Rochester, NY). Equal volumes of diluted cytotoxin were added to the diluted tears and the mixtures were incubated for 1 h at 4 ◦ C. Following incubation, an equal volume (125 ␮l) of a 1% (v/v)

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suspension of washed bovine erythrocytes in TBS CaCl2 buffer was added to each sample, the plates were agitated on an orbital shaker, and then incubated for 3 h at 37 ◦ C. Following incubation the erythrocytes were resuspended and plates were centrifuged at 2500 rpm for 10 min (Beckman TJ-6 centrifuge, Beckman Coulter Inc., Fullerton, CA). Supernatants (200 ␮l samples) were collected and placed in a 96-well microtiter plate for spectrophotometric determination of the optical density (OD) at 455 nm (SpectraMax 250, Molecular Devices Corp., Sunnyvale, CA). Diluted Tifton I cytotoxin and TBS CaCl2 buffer served as respective positive and negative controls. The percent neutralization of each sample was calculated as previously described [9]. If ≥95% neutralization occurred, samples were diluted further until <95% neutralization was observed. A tear neutralizing index (NI) was calculated from the formula: NI =

serum IgG (1:160,000); antigen specific serum IgG (1:400); total tear IgG (1:1000); total tear IgA (1:2000); antigen specific tear IgG and tear IgA (1:10). Wells were then washed twice and 100 ␮l of either sheep anti-bovine IgG (1:100,000) conjugated to horseradish peroxidase (HRP) (Bethyl Laboratories, Inc.) or sheep anti-bovine IgA-HRP (1:35,000; Bethyl Laboratories, Inc.) was added and plates were incubated for 1 h. Following incubation, plates were washed four times and enzyme substrate (Stable Stop ELISA TMB peroxidase substrate; Moss Inc., Pasadena, MD) was added. Reactions were stopped after 30 min with 0.2 N HCl and the optical density (OD) at 450 nm was determined on an automated plate reader (SpectraMax 250). Variations in optical densities of the standard curves generated on each plate to calculate total and antigen specific

[(% neutralization/100) × natural log of the dilution at which < 95% neutralization was observed] total mg tear protein added

Triplicate assays were performed for each sample; the average result from three assays was used as the final tear NI. Serum neutralization assays were performed as described above for tear samples except that the Tifton I cytotoxin was diluted 1:32 before adding it to diluted serum samples. Following the addition of the washed bovine erythrocytes, microtiter plates were incubated at a 45◦ angle at 37 ◦ C for 3 h to allow erythrocytes to sediment in the corners of each well. Following incubation, plates were laid flat and individual wells were scored visually for the presence of hemolysis. The dilution endpoint was defined as the last dilution at which no hemolysis was observed. The geometric mean of three sample endpoints was used as the dilution endpoint. The natural logarithm of the dilution endpoint was used to express the endpoint in statistical analyses. 2.11. ELISA procedures Antigen specific and total serum IgG, tear IgG, and tear IgA were determined by ELISA. The assays were performed in microtiter plates (C-shaped wells; Nunc Immuno Plate Maxisorp surface) at room temperature. For isotype ELISAs, wells were coated for 1 h with affinity purified sheep antibovine IgG or IgA (Bethyl Laboratories, Montgomery, TX) diluted 1:100 in coating buffer (0.05 M sodium carbonate, pH 9.6). For antigen specific ELISAs, wells were coated for 1 hour with the recombinant carboxy terminus of M. bovis cytotoxin (5 ␮g/ml) diluted in coating buffer. Plates were washed twice with 50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0, incubated for 30 min in the same buffer, and then washed two more times. Following the second wash, 100 ␮l of bovine reference serum (Bethyl Laboratories, Inc.) or either diluted calf sera or tear samples were added to each well and plates were incubated for 1 h. Appropriate dilutions of reference serum were made to generate a standard curve and were assayed in duplicate; unknown serum/tear samples were assayed in triplicate at the following dilutions: total

IgG/IgA were corrected using a published formula [30]: corrected OD = [(mean OD of all test day replicates x mean OD of positive control from all assays)/mean test day OD of positive control]. The bovine reference serum served as the positive control for total IgG or IgA isotypes in this calculation. The positive control for the antigen specific IgG or IgA was serum or tears from one calf that was identified during the ELISA optimization to have high antigen specific serum IgG and tear IgG/IgA. The final concentrations of total and antigen specific serum/tear IgG or tear IgA were determined from the slopes of the corrected standard curves. The mean of 3 assays was used to represent the final immunoglobulin concentration of each sample. To correct for differences between groups with respect to antigen specific isotype responses as a result of varying amounts of total immunoglobulin present in each sample, ratios of antigen specific: total immunoglobulin isotype were calculated as follows: serum IgG ratio = the concentration of antigen specific serum IgG (␮g/ml) divided by total IgG (mg/ml); tear Ig ratio = (the concentration of antigen specific isotype Ig (␮g/ml)/total isotype Ig (␮g/ml)) × 100. Differences in immune responses between groups were evaluated by determining mean and percent change in the immune response variables (serum neutralizing titer, tear NI, and serum or tear isotype ratios) from week 0 (prevaccination) to week 7 (4 weeks post-booster). To evaluate vaccine effects on immune response variables while also reducing effects that natural infection would also have on the same variables, nonulcerated calves in the three groups were analyzed. To evaluate the effect that natural infection had on the immune response variables, ulcerated versus nonulcerated calves in the saline group were analyzed. 2.12. Data analysis The cumulative proportion of calves with corneal ulcers was determined each week. Differences between the saline

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and recombinant vaccine groups were evaluated at weeks 12 and 20 by Pearson χ2 analysis. In analyzing differences at these time points, a Bonnferroni adjustment for multiple comparisons was made; here a significance level of P ≤ 0.025 was used to reject the null hypothesis. Differences in immune responses in serum and tears and calf ages were evaluated by independent samples T-tests. Linear regression was used to evaluate associations between variables. In these comparisons, a value of P <0.05 was considered significant. Statistical analyses were performed with SPSS (version 10.0; SPSS Inc., Chicago, IL) and Statview (version 5.0; SAS Institute Inc., Cary, NC) software for windows operating systems. Fig. 1. Cumulative proportion of ulcerated calves over time.

3. Results 3.1. Efficacy of recombinant cytotoxin vaccine for prevention of IBK Throughout the 20 week trial, the cumulative proportion of ulcerated calves was lowest in the recombinant vaccine group (Fig. 1). There were significantly fewer ulcerated calves in the recombinant vaccine group as compared to the saline control group (P = 0.025; Table 1) at week 12, however, by week 20, although the cumulative proportion of ulcerated calves remained lowest in the recombinant vaccine

group, the difference from the saline group was not significant (P = 0.131). The mean number of days until the first ulcer was observed was longer in vaccinates (61.4 ± 8.7 d) than in calves of either control group (saline group: 48.5 ± 4.1 d; adjuvant group: 44.4 ± 5.8 d) although the differences were not significant (Table 1). Although the mean SAM of first ulcers at first observation was largest for the recombinant vaccine group, the difference was not significant. Similarly, no significant difference in peak size of first ulcers, time to healing of first ulcers, or treatment requirements were observed between the three groups.

Table 1 Differences by group in variables assessing vaccine efficacy Groupa

Variable Saline

Adjuvant

Recombinant cytotoxin

Cumulative proportion ulcerated at week 12 Number of calves ulcerated

0.586 17

0.516 16

0.303 10

Cumulative proportion ulcerated at week 20 Number of calves ulcerated

0.586 17

0.548 17

0.394 13

Mean days (±S.E.) to first ulcer N Pb

48.5 (4.1) 17 0.196

44.4 (5.8) 17 0.104

61.4 (8.7) 13

Mean SAM (±S.E.) (cm) for first ulcer N P

0.414 (0.109) 17 0.234

0.398 (0.099) 17 0.186

0.642 (0.160) 13

Peak SAM (±S.E.) (cm) for first ulcerc N P

0.269 (0.067) 10 0.579

0.154 (0.021) 5 0.540

0.206 (0.078) 5

Mean days to heal (±S.E.) of first ulcerc N P

12.6 (2.9) 10 0.347

Proportion requiring treatmentd No. treatments P a b c d

0.31 9 0.364

Sample size: saline group: 29; adjuvant group: 31; recombinant vaccine: 33. The P-value indicated reflects the comparison with the recombinant cytotoxin group. Excludes calves that were treated with florfenicol. Expressed as number of Nuflor treatments/number of calves in group.

11.2 (1.7) 5 0.242 0.48 15 0.600

8.4 (1.4) 5 0.36 12

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Table 2 Mean (±S.E.) changes from week 0 to 7 in variables assessing the effects of a recombinant cytotoxin vaccine on the systemic and local ocular immune responses of nonulcerated calves Variables

Serum neutralizing titer Serum IgG ratio Change (%) Tear neutralizing index Change (%)

Group Saline (N = 17)

Adjuvant (N = 18)

0.94 (0.24)

0.27 (0.18)a

0.08 (0.10) 25.3 3.7 (2.2) 361.4

Recombinant cytotoxin (N = 25) 1.65 (0.15)a b

−0.07 (0.08) 0.3

13.07 (1.46)c 2048.6

0.8 (0.3)d 76.0

6.8 (4.3) 771.2

Tear IgA ratio Change (%)

0.19 (0.19) 98.0

0.13 (0.03)d 43.9

0.06 (0.06) 55.1

Tear IgG ratio Change (%)

0.02 (0.04) 53.5

0.01 (0.03)d 38.9

0.70 (0.14)c 440.0

a b c d

Significantly different from saline group (P < 0.05). Significantly different from adjuvant group (P < 0.001). Significantly different from saline and adjuvant groups (P < 0.001). For tear variables, the adjuvant group sample size was 17 due to inadequate tear sample volume for one calf.

3.2. Systemic and local immune responses following vaccination The mean changes from week 0 to 7 in the serum neutralizing titer, serum IgG ratio, and tear IgG ratio of vaccinated calves were significantly higher in nonulcerated calves that received the recombinant vaccine as compared to nonulcerated calves in either the saline or adjuvant groups (Table 2). Significant differences were not observed between groups in the tear NI or tear IgA ratio, although the tear NI was highest in the vaccinated calves (Table 2). A significant linear relationship (P < 0.001) was found between the change (week 0–7) in serum and tear IgG ratios

amongst week 7 nonulcerated cytotoxin vaccinated calves (see Fig. 2). 3.3. Systemic and local immune responses during natural infection The mean change (week 0–7) in immune response variables in saline control calves were used to examine the effects of naturally occurring IBK on systemic and local immune responses. Calves that were nonulcerated by week 7 had a greater rise in serum neutralizing titer and serum IgG ratio than did calves that were ulcerated by week 7. These differences, however, were not significant (Table 3). Ocular immune responses revealed a similar trend towards greater increases in tear neutralizing index and tear IgG ratio in the nonulcerated calves except for the change in tear IgA ratio which was greatest in ulcerated calves; however, the differences were not significant. Saline control calves that had not developed corneal ulcers by week 7 were significantly older (248.2 ± 5.2 days) than saline control calves that had ulcerated by week 7 (217.5 ± 13.3 days; P < 0.05). The mean week 7 ages of ulcerated and nonulcerated calves were not significantly different for the adjuvant or recombinant vaccine groups. Table 3 Mean (±S.E.) changes from week 0 to 7 in variables assessing systemic and ocular immune responses of ulcerated and nonulcerated saline control calves

Fig. 2. Scatterplot of the week 0–7 changes in the tear IgG ratio vs. serum IgG ratio in calves of the recombinant cytotoxin vaccine group that were nonulcerated at week 7.

Variable

Nonulcerated (N = 17)

Serum neutralizing titer Serum IgG ratio Tear neutralizing index Tear IgA ratio Tear IgG ratio

0.94 (0.24) 0.08 (0.10) 3.7 (2.2) 0.19 (0.19) 0.02 (0.04)

Ulcerated (N = 12) 0.56 (0.23) −0.22 (0.17) −0.2 (1.7) 0.44 (0.27) −0.03 (0.05)

P 0.280 0.137 0.203 0.443 0.409

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4. Discussion In this study, a trend for a reduction in the cumulative proportion of ulcerated calves was observed in calves that received a recombinant M. bovis cytotoxin-ISCOM matrix adjuvanted vaccine. While the difference between vaccinates and controls in the cumulative proportion of ulcerated calves was significant at week 12, it was not significant at week 20; nevertheless, this proportion remained lowest for the recombinant vaccine group throughout the trial. Additional numbers of animals in each group and a higher incidence of IBK in the herd would have increased power to have detected a significant difference between groups; however, it remains unclear whether these factors could have added to the level of significance in our study. Other variables that were used to assess vaccine efficacy included time to development of an ulcer, ulcer size at first observation, peak ulcer size attained of nontreated first ulcers, healing time of first ulcers, and requirements for antibiotic treatment. There were no significant differences in these variables between groups, however, beneficial trends such as longer times to first ulceration and faster healing times were observed in vaccinated calves. To characterize any effect that natural infection had on systemic/local immune responses, we examined immune responses in nonulcerated versus ulcerated saline control calves. Ulcerated saline control calves had a higher tear IgA ratio than nonulcerated saline control calves, an observation that would fit with generation of a local ocular mucosal response following a mucosal infection. Based on the 1 month age difference between the calves that ulcerated by week 7 versus those that did not, previous exposure to M. bovis cytotoxin may have occurred. Although we only enrolled calves with normal corneas, microbiological culturing may have eliminated the inclusion of M. bovis carrier cattle in the study. Prevaccination screening for cytotoxin specific immunoglubulins could also have been performed, however, in earlier studies, asymptomatic carriers had no detectable serum [31] or tear antibodies to M. bovis antigens [32]. In addition, no correlation was found between ocular culture rates of M. bovis and ocular immunity as measured by a passive hemagglutination test using sheep erythrocytes sensitized with crude M. bovis antigens [33]. Because eyes were not cultured in the present study, it was not possible to correlate the type or magnitude of the systemic and local ocular response to presence/absence of M. bovis. The recombinant vaccine used in this study only incorporated the carboxy terminus of the M. bovis cytotoxin. Previously we showed that rabbit antisera against the carboxy terminus (amino acids 643 through 927) could neutralize hemolytic and cytolytic activity of native M. bovis cytotoxin [9]. In this trial, a slightly larger peptide (amino acids 590 through 927) was used in the recombinant vaccine. It is possible that neutralizing epitopes contained at the amino end of the cytotoxin could have improved the level of protection against IBK. In E. coli HlyA, monoclonal antibodies to amino terminal cyanogen bromide cleaved fragments, as well

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as to carboxy terminal fragments neutralize hemolytic activity [34]. In addition to modification of the antigen used in this study, it is also possible that intranasal or intraocular routes of inoculation could improve efficacy of a recombinant cytotoxin vaccine. Previously, a pilus-based M. bovis vaccine was found to be more effective when administered by the aerosol versus the subcutaneous route [35,36]. Disagreement exists in the literature regarding the benefits of systemic versus local ocular immune responses in protecting cattle against IBK. Lacrimal secretions of calves with severe IBK were found to have elevated levels of IgA to crude M. bovis antigens [32]. Although authors of that study were unsure of the role that such secreted antibodies played in IBK protection, it was suggested that local, presumably ocular vaccination may be beneficial. Ocular immune responses to a crude whole cell M. bovis antigen during naturally occurring IBK were then shown to be primarily IgG responses; the authors of that study concluded that tear antibodies did not prevent IBK [37]. Following that study, experimentally infected calves were shown to develop an IgG response in serum and a predominantly IgA response in tears; in that study an ELISA using crude M. bovis antigens was utilized for assaying IgG/IgA [38]. Lacrimal secretory IgA and humoral IgG against a crude M. bovis whole cell extract were later reported to confer resistance to IBK as compared to a humoral IgG antibody response alone [39]. The lack of agreement in the literature as regards the role of ocular immune responses to M. bovis and of its significance in IBK protection is most likely accounted for by differences in the antigen preparations used as well as by lack of adequate controls for total antibody isotypes present in test samples. One previous study that utilized a purified M. bovis hemolysin as antigen in assays to quantitate humoral responses identified specific anti-hemolysin antisera in cattle that were exposed naturally to IBK [26]. In the present study, we attempted to control for fluctuations in total immunoglobulin isotype present in serum and tears by measuring antigen specific and total IgG or IgA and calculating a ratio of antigen specific Ig:total Ig in the analyses. The use of the same purified recombinant protein as the target antigen in the ELISAs also ensured that the responses measured were to the same antigen; the use of such a purified antigen should benefit future studies of systemic and local ocular immune responses to M. bovis infection. The significantly elevated serum and tear IgG ratios and serum neutralizing titers in nonulcerated cytotoxin vaccinated calves, in conjunction with our observation of an overall trend of reduced corneal ulceration in cytotoxin vaccinated calves supports a role for cytotoxin specific IgG in IBK protection. Given the apparent correlation that we observed between ocular antigen specific IgG and systemic antigen specific IgG, as well as from earlier work showing that bovine tear IgG1 is derived from serum [40], it would seem that subcutaneous vaccination using conventional adjuvants could be a reasonable choice for IBK vaccine development. Such a correlation also makes it difficult to define whether systemic or local responses play a greater role in IBK protection. Most

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likely, both are important, and insofar as a relationship exists between systemic and tear IgG in cattle, the two are probably inseparable in terms of which is more important to IBK protection. Nevertheless, the benefit of augmented local ocular cytotoxin specific IgA requires further study. The larger mean size of ulcers in the cytotoxin vaccinated calves could have resulted from IgG mediated complement fixation and subsequent local immune mediated ocular injury following attraction of neutrophils to the site of infection; this mechanism of immune mediated ocular injury could be reduced by adjuvants/routes of inoculation that elicit predominant ocular IgA responses. Results from this study support further investigation into M. bovis cytotoxin based vaccines to prevent IBK.

Acknowledgements Research was funded by UC Davis School of Veterinary Medicine Formula Funds (AH-125 and 4979H), and 1999–2000 Center for Food Animal Health Funds. The authors thank J. Michael Connor, Matthew L. Sween, and Edward Coffin of the University of California Sierra Foothill Research and Extension Center for use of cattle and facilities to complete this study. Assistance from Dr. Steve Gerratt, Judy E. Mihalyi, and Jodi T. Casselman with sample collection and Dr. Erica Dueger with statistical analyses is gratefully acknowledged.

References [1] Henson JB, Grumbles LC. Infectious Bovine Keratoconjunctivitis. I Etiology Am J Vet Res 1960:761–6. [2] Gerhardt RR, Allen JW, Greene WH, Smith PC. The role of face flies in an episode of infectious bovine keratoconjunctivitis. J Am Vet Med Assoc 1982;180(2):156–9. [3] Glass Jr HW, Gerhardt RR. Recovery of Moraxella bovis (Hauduroy) from the crops of face flies (Diptera: Muscidae) fed on the eyes of cattle with infectious bovine keratoconjunctivitis. J Econ Entomol 1983;76(3):532–4. [4] Glass Jr HW, Gerhardt RR. Transmission of Moraxella bovis by regurgitation from the crop of the face fly (Diptera: Muscidae). J Econ Entomol 1984;77(2):399–401. [5] Vogelweid CM, Miller RB, Berg JN, Kinden DA. Scanning electron microscopy of bovine corneas irradiated with sun lamps and challenge exposed with Moraxella bovis. Am J Vet Res 1986;47(2):378–84. [6] Lepper AW, Barton IJ. Infectious bovine keratoconjunctivitis: seasonal variation in cultural, biochemical and immunoreactive properties of Moraxella bovis isolated from the eyes of cattle. Aust Vet J 1987;64(2):33–9. [7] Pugh Jr GW, Hughes DE. Experimental bovine infectious keratoconjunctivitis caused by sunlamp irradiation and Moraxella bovis infection: correlation of hemolytic ability and pathogenicity. Am J Vet Res 1968;29(4):835–9. [8] Beard MK, Moore LJ. Reproduction of bovine keratoconjunctivitis with a purified haemolytic and cytotoxic fraction of Moraxella bovis. Vet Microbiol 1994;42(1):15–33.

[9] Angelos JA, Hess JF, George LW. Cloning and characterization of a Moraxella bovis cytotoxin gene. Am J Vet Res 2001;62(8):1222– 8. [10] Gray JT, Fedorka-Cray PJ, Rogers DG. Partial characterization of a Moraxella bovis cytolysin. Vet Microbiol 1995;43(2-3):183–96. [11] Hoien-Dalen PS, Rosenbusch RF, Roth JA. Comparative characterization of the leukocidic and hemolytic activity of Moraxella bovis. Am J Vet Res 1990;51(2):191–6. [12] Kagonyera GM, George LW, Munn R. Cytopathic effects of Moraxella bovis on cultured bovine neutrophils and corneal epithelial cells. Am J Vet Res 1989;50(1):10–7. [13] Kagonyera GM, George L, Miller M. Effects of Moraxella bovis and culture filtrates on 51Cr-labeled bovine neutrophils. Am J Vet Res 1989;50(1):18–21. [14] Moore LJ, Rutter JM. Attachment of Moraxella bovis to calf corneal cells and inhibition by antiserum. Aust Vet J 1989;66(2):39–42. [15] Ruehl WW, Marrs C, Beard MK, et al. Q pili enhance the attachment of Moraxella bovis to bovine corneas in vitro. Mol Microbiol 1993;7(2):285–8. [16] Annuar BO, Wilcox GE. Adherence of Moraxella bovis to cell cultures of bovine origin. Res Vet Sci 1985;39(2):241–6. [17] Lehr C, Jayappa HG, Goodnow RA. Serologic and protective characterization of Moraxella bovis pili. Cornell Vet 1985;75(4):484–92. [18] Jayappa HG, Lehr C. Pathogenicity and immunogenicity of piliated and nonpiliated phases of Moraxella bovis in calves. Am J Vet Res 1986;47(10):2217–21. [19] Lepper AW, Atwell JL, Lehrbach PR, Schwartzkoff CL, Egerton JR, Tennent JM. The protective efficacy of cloned Moraxella bovis pili in monovalent and multivalent vaccine formulations against experimentally induced infectious bovine keratoconjunctivitis (IBK). Vet Microbiol 1995;45(2-3):129–38. [20] Lepper AW, Elleman TC, Hoyne PA, et al. A Moraxella bovis pili vaccine produced by recombinant DNA technology for the prevention of infectious bovine keratoconjunctivitis. Vet Microbiol 1993;36(1-2):175–83. [21] Lepper AW. Vaccination against infectious bovine keratoconjunctivitis: protective efficacy and antibody response induced by pili of homologous and heterologous strains of Moraxella bovis. Aust Vet J 1988;65(10):310–6. [22] Moore LJ, Lepper AW. A unified serotyping scheme for Moraxella bovis. Vet Microbiol 1991;29(1):75–83. [23] Marrs CF, Ruehl WW, Schoolnik GK, Falkow S. Pilin-gene phase variation of Moraxella bovis is caused by an inversion of the pilin genes. J Bacteriol 1988;170(7):3032–9. [24] Billson FM, Hodgson JL, Egerton JR, et al. A haemolytic cellfree preparation of Moraxella bovis confers protection against infectious bovine keratoconjunctivitis. FEMS Microbiol Lett 1994;124(1):69–74. [25] Nakazawa M, Nemoto H. Hemolytic activity of Moraxella bovis. Nippon Juigaku Zasshi 1979;41(4):363–7. [26] Ostle AG, Rosenbusch RF. Immunogenicity of Moraxella bovis hemolysin. Am J Vet Res 1985;46(5):1011–4. [27] Tabor S, Richardson CC. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci USA 1985;82:1074–8. [28] Nagai K, Thogersen HC. Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli. Meth Enzymol 1987;153:461–81. [29] Coligan JE. Current protocols in immunology, Greene Publishers. Associates and Wiley-Interscience, New York, 1991. v. (loose-leaf). [30] Corbeil LB, Anderson ML, Corbeil RR, Eddow JM, BonDurant RH. Female reproductive tract immunity in bovine trichomoniasis. Am J Reprod Immunol 1998;39(3):189–98. [31] Powe TA, Nusbaum KE, Hoover TR, Rossmanith SR, Smith PC. Prevalence of nonclinical Moraxella bovis infections in bulls as determined by ocular culture and serum antibody titer. J Vet Diagn Invest 1992;4(1):78–9.

J.A. Angelos et al. / Vaccine 23 (2004) 537–545 [32] Nayar PS, Saunders JR. Infectious bovine keratoconjunctivitis. Part II. Antibodies in lacrimal secretions of cattle naturally or experimentally infected with Moraxella bovis. Can J Comp Med 1975;39(1):32–40. [33] Arora AK, Killinger AH, Myers WL. Detection of Moraxella bovis antibodies in infectious bovine keratoconjunctivitis by a passive hemagglutination test. Am J Vet Res 1976;37(12):1489–92. [34] Ji GE, O’Hanley P. Epitopes of Escherichia coli alpha-hemolysin: identification of monoclonal antibodies that prevent hemolysis. Infect Immunol 1990;58(9):3029–35. [35] Misiura M. Estimation of fimbrial vaccine effectiveness in protection against keratoconjunctivitis infectiosa in calves considering different routes of introducing vaccine antigene. Arch Vet Pol 1994;34(34):177–86.

545

[36] Misiura M. Keratoconjunctivitis infectiosa in calves—attempt at elimination by active immunization. Arch Vet Pol 1994;34(34):187–94. [37] Killinger AH, Weisiger RM, Helper LC, Mansfield ME. Detection of Moraxella bovis antibodies in the SIgA, IgG, and IgM classes of immunoglobulin in bovine lacrimal secretions by an indirect fluorescent antibody test. Am J Vet Res 1978;39(6):931–4. [38] Bishop B, Schurig GG, Troutt HF. Enzyme-linked immunosorbent assay for measurement of anti-Moraxella bovis antibodies. Am J Vet Res 1982;43(8):1443–5. [39] Smith PC, Greene WH, Allen JW. Antibodies related to resistance in bovine pinkeye. California Veterinarian; July/August 1989:7–10;18. [40] Pedersen KB. The origin of immunoglobulin-G in bovine tears. Acta Pathol Microbiol Scand [B] Microbiol Immunol 1973;81(2):245–52.