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Duration of immunity against Mycobacterium bovis following neonatal 1
vaccination with BCG Danish: significant protection against infection at 12, but 2
not 24 months 3
4
Thom ML1, McAulay M1, Vordermeier HM2, Clifford, D2, Hewinson RG2, Villarreal-5
Ramos B1, 2 & Hope JC1¥* 6
7
1Institute for Animal Health, Compton, Newbury, Berkshire, RG207NN. 8
2Animal Health and Veterinary Laboratories Agency Weybridge, New Haw, 9
Addlestone, Surrey KT153NB. 10
11
12
*Corresponding author: JC Hope 13
¥Current address: Roslin Institute, University of Edinburgh, Easter Bush, Midlothian, 14
EH259EP, UK. Telephone 0131-6519120 jayne.hope@roslin.ed.ac.uk 15
16
17
Copyright © 2012, American Society for Microbiology. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.00301-12 CVI Accepts, published online ahead of print on 20 June 2012
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Abstract 18
Vaccination of neonatal calves with Mycobacterium bovis Bacille Calmette-Guerin 19
(BCG) induces a significant degree of protection against bovine tuberculosis, caused 20
by infection with virulent M. bovis. In two independent experiments we assessed the 21
duration of the protective immunity induced in calves by neonatal vaccination with 22
BCG Danish. Protection from disease was assessed at 12 and 24 months post-23
vaccination in cattle challenged via the endotracheal route with M. bovis. We also 24
assessed antigen-specific immune responses to assess their utility as correlates of 25
protection. 26
12 months post-vaccination significant reductions in lung and lymph node 27
pathologies were observed compared to non-vaccinated M. bovis challenged control 28
cattle. At 24 months post-BCG vaccination there was a reduction in lung and lymph 29
node pathology scores, and in bacterial burden. However, when comparing 30
vaccinated and control groups, this did not reach statistical significance. Vaccination 31
induced long lived antigen (purified protein derivative, PPD)-specific IFNγ release in 32
whole blood cultures, which remained above baseline levels for more than 20 33
months (approximately 90 weeks). The number of antigen-specific IFNγ secreting 34
central memory T cells present at the time of M. bovis challenge was significantly 35
higher in vaccinated compared with control animals at 12 months post-vaccination 36
but not at 24 months. 37
Vaccination of neonatal calves with BCG Danish induced protective immune 38
responses against bovine TB which were maintained for at least 12 months post-39
vaccination. These studies provide data on the immunity induced by BCG 40
vaccination in calves that could inform vaccination strategies for the control of bovine 41
TB in UK cattle herds. 42
43
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Introduction 44
Bovine tuberculosis (bTB) affects a significant number of cattle in the UK with 45
associated economic and animal welfare concerns. The incidence of bovine TB 46
continues to rise with more than 10% of cattle herds in England currently under TB 47
restrictions. Currently, the diagnosis of bovine TB is by tuberculin skin test combined 48
with ancillary blood tests to detect antigen specific interferon gamma (IFNγ) 49
secretion. Cattle which test positive are subsequently culled from affected herds. 50
However, the control of bTB is complicated by the presence of M. bovis infected 51
wildlife species, including the Eurasian badger, which act as reservoirs of infection. It 52
is clear that improved control strategies are required to reduce the incidence of bTB. 53
Vaccination could become an important element of bovine TB control in the 54
future. Currently, the only licensed vaccine against human TB is Bacille Calmette 55
Guerin (BCG), an attenuated form of Mycobacterium bovis. Alternative approaches 56
to BCG vaccination have been assessed for efficacy in humans and in cattle 57
(reviewed in (6, 23)). However, it has been shown that the most effective strategies 58
that induce protective immunity against bTB are those that include BCG, either alone 59
delivered to calves or as the priming agent in heterologous prime-boost strategies 60
(24, 30). Vaccination of badgers with injectable BCG has been shown to reduce the 61
severity of disease and excretion of M. bovis in experimental studies (8). In field trials 62
BCG vaccination of badgers significantly reduced the number of badgers which 63
tested positive for TB in the Stat-Pak antibody test suggesting that vaccination 64
controlled the progression of disease (8, 20). Vaccination of badgers with BCG has 65
been deployed, on a limited basis, as a control mechanism in England since 2010. 66
Importantly, vaccination of calves with BCG has been shown in a number of studies 67
to induce significant protection against M. bovis infection (5, 7, 16, 34). Following 68
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vaccination with BCG Danish (the strain of BCG licensed for human, and now 69
badger, vaccination) a significant reduction in the number of TB lesions and bacterial 70
burden was observed in the lungs and respiratory tract-associated lymph nodes (16, 71
34) of M. bovis challenged calves. 72
Although a major constraint to the use of BCG as a cattle vaccine is 73
interference with detection of bTB by tuberculin based skin test and IFN-γ detection 74
methods, diagnostic tests utilising M. bovis specific antigens can be employed to 75
distinguish vaccinated from infected animals in DIVA tests (3-4, 9, 29, 31). The 76
implementation of such tests could facilitate the deployment of BCG as a cattle 77
vaccine for bTB control, subject to amendments in EU legislation. 78
An important consideration is the duration of immunity induced by BCG 79
vaccination. We performed studies to determine the duration of protective immunity: 80
calves were vaccinated with BCG and challenged with M. bovis 12 or 24 months 81
post-vaccination. We report herein that reductions in pathology and bacterial burden 82
were evident at 12 and 24 months following vaccination with significant protection 83
from M. bovis challenge evident at 12 months. 84
85
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Materials and Methods 86
Animals and experimental plan 87
Cattle were British Holstein-Friesian calves (Bos taurus) bred at the Institute for 88
Animal Health. The IAH herd has been confirmed free from bovine TB for more than 89
ten years. Two separate experiments, each with groups of age matched vaccinated 90
and control calves were performed. Animals were vaccinated with BCG when aged 91
between 14 days and 29 days of age (experiment 1: median age at vaccination 17 92
days, range 10-27 days; experiment 2: median 25, range 19-29 days). Controls were 93
age matched, non-vaccinated calves. Calves were assigned to groups (9 animals 94
per group) according to age and pre-existing responses to mycobacterial antigens 95
(determined as PPD-specific IFN-γ; see below). Approximately 12 (experiment 1) or 96
24 (experiment 2) months following vaccination, cattle were transferred into a high 97
security ACDP category 3 animal housing unit at AHVLA Weybridge and infected 98
with M. bovis via the endotracheal route. Twelve weeks following infection tuberculin 99
skin tests were performed. The animals were killed one week later and post mortem 100
examinations were performed as described below. The experiments were approved 101
by the local ethics committee according to national UK guidelines. 102
103
Bacteria and calf inoculations 104
BCG Danish (batch numbers 104051C and 106052A; used in experiments 1 and 2 105
respectively) was purchased from Statens Serum Institute (SSI), Denmark and 106
reconstituted immediately prior to use in 1ml Sauton medium. Calves were 107
inoculated subcutaneously in the left shoulder with 0.5ml (5 times recommended 108
human dose) BCG Danish (16). This corresponds to a dose of 1-4 x 106 colony 109
forming units according to the manufacturers specifications. For challenge, M. bovis 110
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(strain AF 2122/97 (13)) was diluted immediately prior to inoculation from frozen 111
stock in 7H9 medium. Cattle were inoculated endotracheally as previously described 112
(30). The dose of M. bovis was confirmed by titration on modified 7H11 agar (12) 113
and determined to be approximately 2 x 103 cfu per calf. 114
115
116
Post-mortem examination and bacteriology 117
Lymph nodes of the head (lateral and medial retropharyngeal, sub-mandibular, 118
parotid) and thorax (caudal and cranial mediastinal, bronchial left and right, cranial 119
tracheo-bronchial), tonsils, nasal and tracheal mucosa and pulmonary lobes were 120
examined for gross lesions following the cutting of 0.5 to 1 cm slices. Macroscopic 121
lesions were scored using the semi-quantitative systems described below (28): (i) 122
Lungs. Lung lobes (left apical, left cardiac, left diaphragmatic, right apical, right 123
cardiac, right diaphragmatic and right accessory) were examined individually. For 124
each lobe, the following scoring system was applied: 0, no visible lesions; 1, no 125
gross lesions, but lesions apparent upon slicing; 2, <5 gross lesions with diameters 126
of <10 mm; 3, >6 gross lesions with diameters of <10 mm, or a single distinct gross 127
lesion with a diameter of >10 mm; 4, >1 distinct gross lesion with diameters of >10 128
mm; 5, gross coalescing lesions. The scores of the individual lobes were added up to 129
calculate the lung score. 130
(ii) Lymph nodes. The severity of the observed gross pathology in individual lymph 131
nodes was scored by use of the following scoring system: 0, no necrosis or visible 132
lesions; 1, small focus (1 to 2 mm in diameter); 2, several small foci, or a necrotic 133
area of at least 5 by 5 mm; 3, multiple necrotic areas of at least 5 by 5mm distributed 134
throughout the node, or one necrotic area affecting >5% of the node. Individual 135
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lymph node scores were added up to calculate the lymph node score. Both lymph 136
node and lung pathology scores were added to determine the total pathology score 137
per animal. All scoring was performed by the same operator for all animals to ensure 138
scoring consistency. Tissues were fixed in 10% formal saline and processed for 139
histological examination following staining with Haematoxylin & Eosin. Tissues with 140
typical lesions of TB evident by microscopy, but no gross lesions, were given a score 141
of 1 in the overall pathology comparison. Tissue samples were frozen at –70oC for 142
subsequent bacteriological examination by titration of tissue homogenates on 143
modified 7H11 agar (12). Colonies per individual lymph node sample were calculated 144
and CFU loads of individual lymph node samples were added up to estimate 145
bacillary content in a given animal. 146
147
Antigens for whole blood assays 148
Purified protein derivatives from M. avium (PPD-A) and M. bovis (PPD-B) were 149
obtained from the Tuberculin production unit at Animal Health and Veterinary 150
Laboratories Agency (AHVLA), Weybridge. For all in vitro assays Weybridge PPDs 151
were used (1mg/ml, 25000 IU/ml). Recombinant ESAT-6 and CFP-10 were as 152
described (9). These antigens are expressed by M. bovis but not by BCG and may 153
be used to discriminate between vaccinated and infected individuals. 154
155
Tuberculin skin tests 156
The single comparative intradermal tuberculin test with avian and mammalian PPD 157
(PPD-A and PPD-B respectively) was by intra-dermal inoculation of 0.1 ml of PPD-A 158
and PPD-B and reactions read 72 hours later. In experiment 1 (12 months vaccination) 159
Weybridge PPD was used (1mg/ml; 25000 IU/ml). For experiment 2 (24 months 160
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vaccination) PPD was obtained from Prionics AG, Switzerland (potency 25000 IU/ml 161
and 30000 IU/ml for PPD-A and PPD-B, respectively). Results were recorded as 162
increase in skin thickness at 72 hours compared to thickness pre-injection and 163
interpreted according to the standard protocol (European Commission Directive 164
64/432/EEC, as amended) (25). 165
166
Immunological assays 167
Blood was collected into heparin (10 U/ml). For cytokine assays 4 ml of blood was 168
incubated at 37oC for 24 h with PPD-A, PPD-B (final concentration of 20μg per ml) 169
diluted in RPMI with 50μg/ml gentamicin. ESAT-6 and CFP-10 were used at a final 170
concentration of 5μg/ml. An equal volume of RPMI with gentamicin was used as 171
control. The supernatant was removed after centrifugation and stored at –20oC until 172
assayed. IFN-γ was assayed by ELISA as described using recombinant bovine IFN-γ 173
as a standard (19). Each sample assayed was measured in duplicate by ELISA; the 174
variability between samples was less than 5%. 175
176
Cultured ELISPOT 177
Secretion of IFN-γ by cultured PBMC was also assessed by cultured ELISPOT, as 178
previously described (36-37). Briefly, PBMC were stimulated in 24 well plates (2 x 106 179
PBMC/ml, 1 ml aliquots) with PPD-B at 10μg/ml. Cells were fed on days 5 and 8 with 180
recombinant bovine IL-2 (on 10 U/ml), and on days 10 and 12 with fresh tissue 181
culture medium. On day 13, 104 cultured cells were added to wells of ELISPOT 182
plates (17), and cultured together with antigen (PPD-A and PPD-B; 10μg/ml). To 183
each well 1 x 103 autologous monocyte derived dendritic cells (15) were added. Cells 184
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were cultured for 24h and spots were developed (17, 37). Results are expressed as 185
spot forming cells (SFC) per 106 PBMC. 186
187
Statistical analyses 188
Analyses were performed using Minitab version 15. The degree of pathology between 189
BCG vaccinated and control calves was compared using the Mann-Whitney test and 190
degree of bacterial colonisation between BCG vaccinated and control calves was 191
compared using the t-test on logarithmically transformed counts. For analysis of central 192
memory antigen specific T cell responses the data for the control groups was pooled 193
and compared to the vaccinated groups in non-parametric ANOVA analyses. P values 194
< 0.05 were considered significant. 195
196
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Results 197
Vaccination with BCG Danish in neonatal calves induces significant protection 198
against M. bovis challenge at 12, but not 24 months post-vaccination 199
In order to establish the duration of immunity induced by neonatal BCG vaccination 200
groups of neonatal calves were vaccinated with BCG Danish within one month of 201
birth. Age matched control calves were not vaccinated. Approximately 12 months 202
(week 54) or 24 months (week 112) later calves were challenged endotracheally with 203
M. bovis. The protective efficacy of BCG vaccination against disease was assessed 204
by post mortem examinations performed 12 weeks post challenge in all animals 205
(Tables 1 & 2). None of the animals showed clinical signs of disease. Although the 206
extent of lesions varied between animals, as reflected by the lesion scores (Table 1), 207
in each case lesions were primarily restricted to the respiratory tract-associated 208
lymph nodes and lungs. 209
210
In animals infected with M. bovis 12 months post-vaccination (Table 1), lesions 211
typical of TB were observed in all but one of the non-vaccinated controls, in both 212
lymph nodes and lung. Two animals in the BCG vaccinated group had no evidence 213
of lesions. BCG Danish conferred significant protection against M. bovis challenge 214
with reduced total gross pathology (P = 0.0373) and significantly reduced lung 215
pathology scores (P = 0.0303). Lymph node pathology scores were also 216
substantially reduced in vaccinated animals although this reduction did not quite 217
reach statistical significance (P = 0.0685). 218
219
Lesions typical of TB were observed in all of the animals challenged 24 months post-220
vaccination irrespective of their vaccination status (Table 2). Although there was a 221
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tendency towards lower lesion scores within the BCG vaccinated group no significant 222
difference in overall pathology between BCG vaccinated and control cattle was 223
observed (p = 0.1428). No statistically significant difference was observed in lymph 224
node (P = 0.3473) or lung (P= 0.1304) pathology scores between BCG vaccinated 225
and control animals. Overall, although an approximately 20 percent reduction in 226
lesion score was evident in the BCG vaccinated animals (Table 2) this was not a 227
statistically significant decrease. 228
229
BCG vaccination of neonatal calves reduces the level of bacterial colonisation 230
Neonatal BCG vaccination reduced the severity of disease following M. bovis 231
infection. In order to assess whether this was associated with effects on bacterial 232
burden we measured numbers of bacteria present within the respiratory tract 233
associated lymph nodes. Bacteriological examination of tissues indicated that M. 234
bovis was present in the majority of tissues with gross lesions in both experiments 235
(Tables 1 and 2). In cattle challenged 12 months post-vaccination (Table 1) 3/9 236
animals in the BCG vaccinated group had no viable M. bovis cultured from tissues. 237
However, overall there was no significant difference in the number of viable M. bovis 238
(cfu/LN) between control and vaccinated cattle (P = 0.189). In the cattle challenged 239
24 months post-vaccination (Table 2), viable bacteria were isolated from the tissues 240
of all of the animals and no significant differences were observed between the BCG 241
vaccinated and non-vaccinated control animals (P = 0.733). 242
243
244
245
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Skin test reactivity in BCG vaccinated and control calves following M. bovis 246
challenge 247
Reactions to PPD in the skin test were observed in the majority of animals 12 weeks 248
after challenge with M. bovis (Tables 1 and 2). Visible local clinical signs and 249
increases in comparative skin thickness (skin test thickness response PPD-B minus 250
PPD-A) ranging from 7 to 63 mm were recorded. Relatively low responses to PPD-A 251
were noted in these animals. 252
253
In experiment 1 (animals challenged with M. bovis at 12 months post-vaccination, 254
Table 1), one non vaccinated animal and two BCG vaccinated animals showed no 255
skin test reactivity. These animals also had no visible lesions or viable M. bovis 256
present in tissues. However, 7/9 BCG vaccinated and 8/9 control animals would be 257
classified as reactors according to the standard interpretation of the skin test. There 258
were no significant differences between the groups (P= 0.112). 259
260
All of the animals challenged at 24 months post-vaccination had significant skin test 261
reactivity (Table 2). There were no significant differences between the groups (P = 262
0.266) and each of the animals would be classified as reactors according to the 263
standard interpretation of the skin test. 264
265
BCG Danish induces long lived antigen-specific IFN-γ responses in whole blood 266
cultures 267
We measured antigen specific IFNγ secretion in BCG vaccinated and control calves 268
throughout the experiment to monitor the course of the immune response. Calves 269
were vaccinated with BCG Danish and challenged with M. bovis at 12 months 270
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(experiment 1: Figure 1a, b, c) or 24 months (experiment 2: Figure 1d, e, f) post-271
vaccination. 272
In experiment 1 vaccination with BCG Danish induced PPD-B specific immune 273
responses which were evident at 2 weeks post vaccination (Figure 1a). Although the 274
PPD-B specific IFN-γ response was reduced after 6 weeks post-vaccination, this was 275
above the level of PPD-B specific IFN-γ observed in the control group for the 276
duration of the vaccination study (52 weeks). No PPD-B specific IFNγ was observed 277
in the control calves at any time point (< 500pg/ml, data not shown). 278
279
Post M. bovis challenge, PPD-B specific immune responses in whole blood cultures 280
were observed from 4 weeks in both control (Figure 1b) and vaccinated (Figure 1c) 281
animals. No differences between vaccinated and control groups were observed in 282
PPD-B specific IFN-γ at any time point post M. bovis challenge. 283
284
In experiment 2 similar early kinetics of PPD-B-specific IFNγ induction were 285
observed with the peak response evident between 2-8 weeks post-vaccination. 286
These responses remained generally above the pre-vaccination levels up to 90 287
weeks post vaccination (with the exception of weeks 32, 34, 64 and 66; Figure 1d). 288
289
Post-M. bovis challenge, ESAT-6/CFP-10 specific IFNγ secretion was observed in all 290
animals irrespective of vaccination status. In the animals challenged at 12 months 291
(Figure 2), higher responses were observed in the control group (closed symbols) 292
from 4 weeks to 8 weeks following infection. No differences in reactivity to ESAT-293
6/CFP-10 were observed between control and vaccinated animals challenged at 24 294
months. 295
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The frequency of antigen-specific IFN-γ-secreting memory cells present at the time of 296
challenge correlates with protective immunity 297
Detection of IFNγ secreting central memory T lymphocytes in cultured ELISPOT 298
assays has previously been shown to predict protection in BCG vaccinated calves 299
(16, 36) and was used herein to assess whether the detection of memory T cells at 300
12 or 24 months post vaccination was also predictive of efficacy. At 12 months 301
(Figure 3) or 24 months post-vaccination (data not shown), immediately prior to M. 302
bovis challenge, PBMC were isolated from all calves and cultured in the presence of 303
antigen and IL-2 to establish short-term T cell lines. Following 2 weeks of culture, the 304
presence of central memory T cells secreting IFN-γ was assessed by ELISPOT. The 305
frequency of PPD-B-specific SFC (PPD-B minus PPD-A) was calculated. The data 306
for the control animals was pooled for analysis as no significant differences were 307
noted between the two control groups. 308
309
Twelve months following vaccination of cattle with BCG Danish (Figure 3; black bar) 310
antigen specific IFN-γ-expressing central memory T lymphocytes were significantly 311
higher in number than those observed in non-vaccinated control animals (Figure 3; P 312
= 0.02). By contrast, the number of PPD-B specific IFN-γ-expressing central memory 313
T cells present in cattle 24 months post-vaccination was not significantly different 314
from that observed in control animals. 315
316
317
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Discussion 319
Bovine tuberculosis is an economically important disease of increasing incidence in 320
UK cattle herds. Disease control is currently through the skin test and slaughter 321
policy, and ancillary blood tests detecting tuberculin induced cell mediated immune 322
responses. The presence of the causative agent of bovine TB, Mycobacterium bovis, 323
in wildlife reservoirs impacts significantly on the effectiveness of disease control. 324
Hence, additional control tools are required to reduce the incidence of bovine TB in 325
areas of endemic bTB incidence. An important mechanism for control is likely to be 326
vaccination. As an attenuated form of M. bovis, BCG shares high homology with the 327
virulent strain and therefore vaccinated cattle also show skin test reactivity to 328
tuberculin (PPD-B) and also secrete antigen-specific IFNγ in blood based diagnostic 329
tests. However, despite numerous vaccine candidates being trialled in cattle for 330
efficacy against M. bovis infection, the most successful strategies are those which 331
include BCG either as a single vaccine or as the priming agent in heterologous 332
prime-boost regimens (reviewed in (6, 33)). Thus, differentially expressed antigens 333
between M. bovis and BCG, such as ESAT-6, CFP-10 and other RD1 gene 334
products, have been identified and used in diagnostic assessments to provide the 335
capacity for differentiating infected from vaccinated animals (DIVA) (reviewed in 336
(32)). 337
Vaccination of calves with BCG is associated with significant protective 338
efficacy against virulent challenge with M. bovis (5, 7, 16-17, 34). In these studies, 339
vaccination within the first four weeks of life was shown to induce significant 340
reductions in disease associated parameters including lesion (pathology) scores and 341
bacterial burden within the respiratory tract tissues. Protective efficacies of 342
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approximately 70 percent were reported with a significant number of animals 343
completely protected from disease. 344
In experimental studies of BCG vaccination of neonatal calves (5, 7, 16-17, 345
34), there was a relatively short duration between vaccination and infectious 346
challenge with M. bovis: on average a vaccination period of 3 months was utilised. 347
Given that dairy cows have an average lifespan on farm of ≥3 years we sought to 348
determine whether a single vaccination of calves would give long-lasting protection 349
or, to define at which time point protective immunity wanes. This would enable 350
determination of timescales for enhancing immunity through heterologous boosting 351
or homologous re-vaccination. 352
In humans, neonatal vaccination with BCG has a significant impact on 353
childhood TB; however a decline in BCG efficacy was reported over time/with age. 354
These studies imply that relatively short duration of protection induced by BCG 355
vaccination may account, at least in part for the variable efficacy of BCG vaccination 356
that has been reported in human populations (10, 14). In mice, the duration of BCG 357
induced immunity was shown to be less than 40 weeks with a significant reduction in 358
protective efficacy observed between 20 and 40 weeks post-vaccination (26). 359
Herein, we showed that vaccination of calves induced protective immunity 360
against M. bovis challenge and that this was maintained for at least 12 months 361
following vaccination. We observed a similar protective efficacy at 12 months to that 362
previously reported at 3 months post-neonatal vaccination (16). However, the 363
protective immunity had waned by 24 months. Thus, although there was a reduction 364
in each of the parameters assessed in cattle challenged two years after vaccination 365
(lung and lymph node pathology scores and bacterial burden), with the relatively 366
small group sizes utilised in our studies, this did not reach statistical significance. 367
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However, this could potentially have a positive impact on disease control in larger 368
cattle herds by reducing the overall level of disease and numbers of bacilli present. 369
It should also be noted that experimental challenge whereby ~103 M. bovis 370
are placed directly into the respiratory tract is a more aggressive challenge than 371
natural exposure whereby transmission occurs primarily through droplet inhalation of 372
relatively small numbers of bacilli. Interestingly, in a recent field study in Ethiopia 373
where calves were vaccinated with BCG, Ameni et al showed that immunity against 374
naturally transmitted M. bovis infection was long-lived, with duration of at least 23 375
months (1). In these studies a significant proportion of completely protected cattle 376
with no visible lesions at post-mortem examination were observed. In similar studies 377
performed in Mexico (22) the duration of BCG induced immunity against bTB was 378
reported to wane at around 12 months of age, in line with our experimental studies. 379
However, consideration will need to be given to the likelihood that boosting of 380
immunity will be required. In studies where short-interval (6 weeks) homologous 381
boosting of BCG induced immunity was assessed no positive effect on the outcome 382
of infectious challenge was observed (7). However, the effects of homologous BCG 383
boosting or re-vaccination with longer prime-boost intervals (e.g. 1 year post initial 384
BCG vaccination) has not yet been assessed but could stimulate longer duration 385
protection as the initial immunity starts to wane. Heterologous prime boost 386
strategies, utilising viral vectors such as human adenovirus or MVA which express 387
immuno-dominant antigens such as Ag85A, have been shown to be superior to BCG 388
alone in protecting cattle from M. bovis (30) and are currently in Phase IIB clinical 389
trials in humans for vaccination against M. tuberculosis infection (23-24). The 390
capacity for these strategies to boost BCG induced immunity for life-long immunity 391
for cattle should be assessed. 392
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Defining predictors of protective immunity is an essential part of vaccine 393
research. The capability to determine whether vaccines bestow protective immune 394
responses prior to infectious challenge will enable more high-throughput studies 395
prioritising candidates for efficacy studies. This will reduce not only the timescales for 396
vaccine evaluation but will significantly reduce the cost of vaccine studies and 397
reduce the severity of animal procedures. A number of candidate immunological 398
predictors have been identified in TB vaccine studies. In studies of neonatal BCG 399
vaccination we showed that protective efficacy was correlated with the number of 400
PPD-B specific central memory T cells (Tcm) present at the time of M. bovis 401
challenge (16), in line with previous studies of vaccine induced protection from bTB 402
(30, 36). Confirming this observation, we showed herein that when measured 12 403
months post-vaccination, the numbers of Tcm determined by cultured ELISPOT 404
were higher in vaccinated compared to control animals and this corresponded to 405
protection from infection overall. However, by 24 months post-vaccination we were 406
unable to show significant difference in the numbers of Tcm between vaccinated and 407
control cattle. Significantly increased numbers of PPD-B specific Tcm in vaccinated 408
cattle are thus only present in groups of cattle where protective immunity is observed 409
suggesting this may be a predictive correlate of BCG induced protection. 410
The relationship of IFNγ secretion to protective efficacy is complex – whilst it 411
is clear that successful vaccination requires the induction of IFNγ; detection of IFNγ 412
alone is not a sufficient biomarker of protection (18, 27, 33). We observed increased 413
antigen-specific IFNγ secretion for up to 90 weeks following vaccination but could not 414
correlate this with the observed levels with protection from M. bovis challenge. In 415
studies of the duration of BCG induced immunity in mice, IFNγ responses of CD4+ T 416
cells to M. tuberculosis antigens were maintained despite a loss of protection 417
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between 20 and 40 weeks post-vaccination (26). More complex measurement of 418
polyfunctional T cells has revealed that multi-parametric responses of lymphocytes 419
are required for protective immunity against M. tuberculosis in humans and mice (2, 420
11, 21). Recent studies in cattle have established methods for the measurement of 421
multifunctional T cells expressing IFNγ, IL-2 and TNFα in M. bovis infection (35) and 422
these may now be applied to vaccine studies to facilitate the definition of predictors 423
of vaccine success. Application of such techniques to future vaccination studies in 424
neonatal calves may be useful in defining predictors and correlates of protection. 425
In summary, we have shown that BCG vaccination of calves induces a 426
significant degree of protective immunity which lasts for at least 12 months but that 427
this wanes by 24 months post-vaccination. These results will inform future studies of 428
the use of BCG vaccination in conjunction with strategies for boosting immunity, or 429
re-vaccination for longer term protection from M. bovis infection. 430
431
432
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Acknowledgements 433
This work was supported by grants from the Department for the Environment, Food 434
and Rural Affairs (DEFRA) UK. We gratefully acknowledge the staff of the IAH and 435
VLA animal facilities for care of the cattle, and members of the TB group at VLA for 436
assistance with necropsy and bacteriological evaluations. Statistical advice was 437
received from Professor G Gettinby at Strathclyde University 438
JC Hope, HM Vordermeier and RG Hewinson are Jenner Institute Investigators. 439
440
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578 579
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Figure Legends 581
Figure 1: Antigen-specific IFNγ secretion in whole blood cultures 582
IFNγ release from bovine whole blood cultures stimulated with PPD-A (open 583
squares), PPD-B (closed squares), medium (open circles) from animals: (a) 584
Vaccinated with BCG Danish; (b) Non-vaccinated control animals challenged with M. 585
bovis at 12 months (week 54); (c) 586
Vaccinated with BCG Danish at week 0 and challenged with M. bovis at 12 months 587
(week 54); (d) Vaccinated with BCG Danish [experiment 2]; (e) Non-vaccinated 588
control animals challenged with M. bovis at 24 months (week 114); (f) Vaccinated 589
with BCG Danish at week 0 and challenged with M. bovis at 24 months (week 114). 590
IFNγ levels are expressed as group mean values (± SE) plasma concentrations 591
(pg/ml). 592
593
Figure 2: IFNγ released following stimulation of whole blood with an ESAT-594
6/CFP-10 cocktail 595
IFNγ secretion was assessed post M. bovis challenge in BCG vaccinated (open 596
symbols) and non-vaccinated control animals (closed symbols). Animals were 597
challenged with M. bovis 12 months post-vaccination and blood samples were 598
assessed bi-weekly. IFNγ levels are expressed as group mean values (± SE) plasma 599
concentrations (pg/ml). 600
601
Figure 3: Frequency of antigen specific IFNγ secreting T lymphocytes present 602
at the time of M. bovis challenge 603
Twelve months (black bar) or 24 months (grey bar) post BCG vaccination, memory 604
cell responses were assessed by cultured ELISPOT. The responses in control 605
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animals were also assessed (open bar): these were not significantly different at 606
either time point and the data were therefore pooled for analysis. The number of 607
PPD-B specific IFN-γ SFC present within PBMC was significantly higher in 608
vaccinated animals at 12 months post-vaccination, but not at 24 months when 609
compared with control, non-vaccinated animals. Mean +/- SD is shown 610
(n = 9 for vaccinated groups; n = 18 for controls). 611
*p < 0.05. 612
613
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Experiment 1: Weeks Post-BCG vaccination
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104
Experiment 2: Weeks Post-BCG vaccination
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Experiment 1: Weeks post-M. bovis challenge Experiment 2: Weeks post-M. bovis challenge
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Controls
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Table 1: Pathology scores and bacteriological counts in animals infected with M. bovis 12 months post vaccination ‡Skin test thickness was recorded at day 0 and day 3 following inoculation with PPD-A and PPD-B. PPD B specific increases in skin thickness at 72 hours are shown.
Control animals
Gross pathology Scores
Animal ID Total LN score Total lung score Total path score CFU/LN sample Skin test B-A‡
402340 14 16 30 587000 47
602342 20 6 26 1142000 53
502348 19 19 38 128400 27
302353 9 7 16 483480 25
502355 11 4 15 47000 26
401361 15 13 28 990400 37
102365 2 5 7 11400 27
302367 8 5 13 92200 36
602370 0 0 0 0 0
Median 11 6 16 Mean: 386875 Mean: 30.9
BCG
Vaccination
Gross pathology Scores
Animal ID Total LN score Total lung score Total path score CFU/LN sample Skin test B-A‡
502341 10 3 13 842020 49
702343 8 0 8 29740 22
702350 1 0 1 0 7
402354 12 0 12 46060 18
302360 9 7 16 310020 8
202366 7 4 11 77400 30
502369 0 0 0 0 0
102372 0 7 7 7260 32
402375 0 0 0 0 0
Median 7 0 8 Mean: 145833 Mean: 18.4
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Table 2: Pathology scores and bacteriological counts in animals infected with M. bovis 24 months post vaccination ‡Skin test thickness was recorded at day 0 and day 3 following inoculation with PPD-A and PPD-B. PPD B specific increases in skin thickness at 72 hours are shown.
Control animals
Gross pathology Scores
Animal ID Total LN score Total lung score Total path score CFU/LN sample Skin test B-A‡
C09-5134 4 7 11 2400 40
C09-5135 4 6 10 1753 18
C09-5136 5 4 9 62700 63
C09-5137 4 4 8 8297 27
C09-5138 3 5 8 467 18
C09-5139 10 5 15 79020 17
C09-5140 7 10 17 53500 57
C09-5141 10 7 17 47187 21
C09-5142 5 5 10 2167 28
Median 5 5 10 Mean: 28610 Mean: 32.1
BCG
Vaccination
Gross pathology Scores
Animal ID Total LN score Total lung score Total path score CFU/LN sample Skin test B-A‡
C10-6123 4 4 8 1813 27
C10-6124 3 4 7 45900 20
C10-6125 5 4 9 47027 10
C10-6126 9 4 13 5040 30
C10-6129 3 4 7 2373 15
C10-6130 8 9 17 11420 28
C10-6127 7 7 14 16260 38
C10-6128 0 5 5 1040 45
C10-6131 3 3 6 4147 16
Median 4 4 8 Mean: 15002 Mean: 25.4
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