downloaded from on may 29, 2020 by guest · 92 between 14 days and 29 days of age (experiment 1:...

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 [email protected] 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

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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

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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

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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

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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

580


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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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104

Experiment 2: Weeks Post-BCG vaccination

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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



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



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



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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