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The Cross-species Mycobacterial Growth Inhibition Assay (MGIA) Project 2010-2014 1

2

Michael J. Brennan1+

, Rachel Tanner2*+

, Sheldon Morris3, Thomas J. Scriba

4, Jacqueline M. 3

Achkar5, Andrea Zelmer

6, David Hokey

1, Angelo Izzo

7, Sally Sharpe

8, Ann Williams

8, Adam 4

Penn-Nicholson4, Mzwandile Erasmus

4, Elena Stylianou

2, Daniel F. Hoft

9, Helen McShane

2, 5

Helen A. Fletcher6

6

7

1Aeras, Rockville, MD, 20850 USA;

2The Jenner Institute, University of Oxford, Oxford, OX3 8

7DQ UK; 3Center for Biologics, Evaluation and Research, Food and Drug Administration, 9

20892 USA; 4South African Tuberculosis Vaccine Initiative and Division of Immunology, 10

Department of Pathology, University of Cape Town, 7925 South Africa;

5Departments of 11

Medicine, Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, 12

10461 USA; 6Department of Immunology and Infection, London School of Hygiene & 13

Tropical Medicine, London, W1CE 7HT UK; 7Colorado State University, Fort Collins, 14

Colorado, 80523 USA; 8Public Health England, Porton Down, Salisbury, SP4 0JG UK; 15

9Saint Louis University, St. Louis, MO, 63104 USA. 16

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*Corresponding author: Rachel Tanner. Email: [email protected] 18

+ denotes shared first authorship 19

Keywords: Mycobacterial Growth Inhibition Assay (MGIA); Tuberculosis; Correlates of 20

immunity; Vaccines 21

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Abstract 23

The development of a functional biomarker assay in the tuberculosis (TB) field would be 24

widely recognized as a major advance in efforts to efficiently develop and test novel TB 25

CVI Accepted Manuscript Posted Online 12 July 2017Clin. Vaccine Immunol. doi:10.1128/CVI.00142-17Copyright © 2017 Brennan et al.This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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vaccine candidates. We present preliminary studies using mycobacterial growth inhibition 26

assays (MGIAs) to detect a BCG vaccine response across species, and extend this work to 27

determine if a standardized MGIA can be applied in characterizing new generation TB 28

vaccines. The comparative MGIA studies reviewed here aimed to evaluate robustness, 29

reproducibility, and ability to reflect in vivo responses. In doing so, they have laid the 30

foundation for the development of an MGIA that can be standardized and potentially 31

qualified. The major challenge ahead lies in better understanding the relationship between in 32

vivo protection, in vitro growth inhibition and the immune mechanisms involved. The final 33

outcome would be an MGIA that can be used in confidence in TB vaccine trials. We 34

summarize data arising from this project, present a strategy to meet the goals of developing a 35

functional assay for TB vaccine testing, and describe some of the challenges encountered in 36

performing and transferring such assays. 37

38

Introduction 39

The TB vaccine field is severely hampered by the lack of defined immune parameters that 40

correlate with vaccine efficacy in human studies. There has been recent progress, such as the 41

finding that antigen-specific IFN-γ ELISpot responses correlate with reduced risk of 42

developing disease in BCG-vaccinated South African infants[1]. However, heterogeneous 43

BCG vaccine responses are observed which may be associated with the immune environment 44

at the time of vaccination, including the proportion of monocytes and activated HLA-DR+ 45

CD4+ T cells[1, 2]. Development of a validated functional ‘biological response’ assay 46

capable of assessing the potential of a vaccine-induced immune response to protect against 47

Mycobacterium tuberculosis (M.tb) infection or TB disease would represent a major advance 48

in the effort to develop TB vaccines. Such an assay would circumvent the need to identify or 49

fully understand the specific aspects of the immune response contributing to this effect. 50


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Mycobacterial growth inhibition assays (MGIAs) represent one such clinically relevant 51

approach. Using whole blood or peripheral blood mononuclear cells (PBMCs), MGIAs offer 52

an unbiased measure of the vaccine-induced antigen-specific immune response, taking into 53

account the ability of this response to function in its respective immune environment. They 54

also have the potential to shorten the time-frame and resources required to evaluate a new TB 55

vaccine in preclinical studies. 56

The MGIA project brought together an international group of scientific experts to work on 57

growth inhibition assays from 2010 to 2014. Participants included researchers from TB 58

endemic regions such as Cape Town, South Africa; as well as non-endemic areas such as the 59

UK and Washington DC, Saint Louis and Colorado in the US. 60

61

Goals of the MGIA project and assay selection 62

The MGIA project was initially supported by a World Health Organization (WHO) working 63

group, and was viewed as the follow-up to a whole blood ELISA assay that the WHO had 64

previously recommended[3]. The aim was to develop a functional assay that could be applied 65

in all clinical trials of TB vaccine candidates, thus aiding the identification of correlates of 66

immunity. Although several MGIAs were described in the literature previously (recently 67

reviewed in [4]), little work had been done on qualifying an assay that could be transferred 68

across laboratories using a standardized reproducible method for vaccine assessment. This 69

was a major goal of the project, funded by Aeras and the US Food and Drug Administration 70

(FDA). Prior to 2010, most MGIA development had focused on whole blood systems and had 71

shown promise[5-10]. However, especially given the logistical challenges associated with 72

real-time processing of fresh blood in clinical vaccine trials, in these studies, efforts were 73

made to assess the potential of using cryopreserved PBMC and to compare the outcomes with 74

those from whole blood. Other goals included determining the effect of M.tb infection status 75


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on mycobacterial growth inhibition, comparing MGIA responses induced by wild-type BCG 76

and a novel recombinant BCG vaccine, and investigating the induction and functional role of 77

antibodies. The specific early goals are outlined in Table 1. 78

Although four previously-published MGIAs were considered for initial evaluation, a whole 79

blood assay based on the methods of Wallis et al. was selected as a focus for this project, 80

using the BACTEC MGIT system to quantitate mycobacterial growth[5] (henceforth referred 81

to as the ‘direct MGIA’). The rationale for this decision included the relative simplicity of the 82

assay and the use of standardized reagents and equipment that are readily available in many 83

clinical TB laboratories. Furthermore, the assay had previously been used to demonstrate 84

anti-mycobacterial activity of bactericidal drugs that correlated with sterilizing activity 85

observed during in vivo therapy[5, 11]. The BACTEC MGIT system has several advantages 86

over traditional colony-forming unit (CFU) based methods of mycobacterial quantification, 87

as it utilizes oxygen depletion as a detection method which is sensitive not only to the number 88

of viable bacteria, but also their rate of metabolism and growth. It is unaffected by clumping 89

and does not require serial dilutions, providing an accurate computer-generated read-out 90

based on validated technology. 91

It was also agreed that an adaptation of this assay using cryopreserved PBMC be developed 92

and evaluated for comparison with whole blood. It is widely accepted that the cellular 93

immune response is central to protection against M.tb, and although innate whole blood 94

components such as neutrophils are known to play a role in the host response, they may be 95

less relevant when considering a vaccine-induced effect on adaptive immunity. Furthermore, 96

use of cryopreserved cells could aid in transferability of the assay to different trial sites, 97

eliminating the need to evaluate samples in real-time using fresh material. Rather, cells could 98

be stored and run in the future which is not only logistically simpler, but would negate the 99

need for a BACTEC MGIT machine at every site. Finally, the ability to use cryopreserved 100


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cells would enable additional retrospective study of samples from historical clinical vaccine 101

trials for validation and exploratory work. A schematic and description of the method for the 102

direct MGIA assay is provided in Figure 1. 103

The studies involved in this project are presented chronologically; work began using samples 104

from clinical studies (as the ultimate goal was development of a human assay), but the 105

challenges encountered resulted in a decision to focus on preclinical adaptations in the latter 106

stages. One of the greatest impediments to initial MGIA developmental efforts using human 107

samples was the lack of availability of sufficiently large single whole blood or PBMC 108

samples from recently BCG vaccinated volunteers. This was required for assay optimization 109

and assessment of reproducibility between laboratories. Using animal samples such as mouse 110

splenocytes, however, it was possible to overcome this issue as 50-100 million splenocytes 111

may be isolated from each mouse. Furthermore, the ability to select age- and sex-matched 112

naïve inbred animals removes a number of potential sources of variability found in human 113

clinical trials. Animal models also provide the opportunity for biological validation by 114

correlating assay outcome with protection from in vivo challenge with pathogenic M.tb (see 115

below). 116

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Early optimization work 118

BCG was selected as a surrogate for M.tb in the majority of these studies to avoid the need 119

for Biosafety level 3 (BSL3) facilities. It has previously been established that measures of 120

growth inhibition of BCG correlate with those of M.tb H37Rv in a murine MGIA[12], and 121

this was confirmed in the direct MGIA (Figure 2A). Early experiments using the direct 122

PBMC MGIA assay indicated high intra-assay variability between replicate cultures. This 123

was addressed by considering various aspects of assay preparation, the culture period and 96-124

hour processing. It was determined that the standard antibiotic supplement (penicillin and 125


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streptomycin) in culture medium should not be used at any stage of sample processing after 126

thawing, due to the effect of artificially reducing the inoculum[13]. However, this altered the 127

multiplicity of infection (MOI) such that the vaccine effect was overwhelmed. A smaller 128

inoculum resulted in increased intra-assay variability. To maintain the same initial inoculum 129

volume, a lower MOI was achieved by using greater cell concentrations, and although this 130

did increase reproducibility, a balance had to be drawn given the limiting factor of cell 131

number in many clinical trials. Experiments altering the MOI in the direct splenocyte MGIA 132

have been described[14]. Once an MOI was identified, variability in the inoculum itself was 133

addressed by comparing different de-clumping methods such as vortexing, filtering and 134

sonicating. Vortexing with glass beads resulted in low intra-assay variability while retaining a 135

high recovery of CFU. Other variables examined included BCG strain[14], culture rotation 136

methods and lysis agents. Variability between sites in whole blood MGIA performance has 137

yet to be defined, but could result from factors such as differences in blood anticoagulants 138

and inoculum strain. 139

140

Human MGIA Studies 141

1) BCG vaccination and revaccination 142

A clinical trial of BCG vaccination and revaccination in UK adults was conducted at the 143

University of Oxford[13]. The direct PBMC MGIA detected a significant improvement in 144

mycobacterial growth inhibition following primary BCG vaccination, consistent with 145

epidemiological data on the efficacy of BCG in this population. No improvement in 146

mycobacterial growth inhibition was observed following BCG revaccination using either the 147

whole blood or PBMC assay[13]. This is in contrast to a previous study in US volunteers, in 148

which the direct whole blood MGIA, as well as the primary lymphocyte MGIA (effector 149

lymphocytes added to infected autologous monocytes[9]) and secondary lymphocyte MGIA 150


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(using antigen expanded T cells[6]), detected enhanced mycobacterial growth inhibition after 151

BCG revaccination[7]. 152

In the Oxford study, the direct PBMC MGIA demonstrated a stronger primary vaccine effect 153

and greater reproducibility over repeated baseline bleeds compared with whole blood[13]; 154

most likely due to the processing of longitudinal PBMC samples in one batch. This is not an 155

option for fresh blood which may suffer from batch-effects including those associated with 156

different vials of mycobacterial inoculum. However, this was not the case in other studies, 157

such as the AERAS-422 vaccine trial at Saint Louis University described below, in which the 158

direct whole blood MGIA proved less variable among subjects included in each vaccinated 159

group compared to primary, secondary or titrated PBMC MGIAs[15]. 160

A study was also conducted in South Africa, comparing 10 week old infants who were BCG 161

vaccinated at birth with older adults. Infants demonstrated enhanced ability to inhibit growth 162

of BCG SSI (but not M.tb H37Rv) in the direct whole blood MGIA compared with adults 163

(Figure 2B). This data suggest that BCG vaccination may induce preferential growth 164

inhibition of BCG even in newborns. 165

2) Immune correlates of risk study 166

Recently, Fletcher et al. have reported the findings of an immune correlates of risk study in 167

BCG vaccinated infants in South Africa[1]. In this population, BCG-specific IFN- ELISpot 168

responses and levels of Ag85A-specific IgG antibodies measured at 4-6 months of age were 169

associated with a reduced risk of developing TB disease over the next 3 years of life[1]. 170

However, ability to control mycobacterial growth in the direct PBMC MGIA was not 171

associated with reduced risk of TB disease. This could be due to the very low frequency of 172

BCG specific IFN- secreting T-cells (median less than 50 SFC/million PBMC) and the fact 173

that a small number of PBMC were used in the assay (it has since been established that the 174

direct MGIA is more robust when greater numbers of PBMC are used). Furthermore, 175


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autologous serum was not used in the assay and it would therefore not have measured the 176

contribution of Ag85A specific IgG, or any other antibody, to growth inhibition[1]. This was 177

also prior to subsequent further optimization of the assay. 178

3) Comparing wild-type BCG and a new recombinant BCG vaccine 179

Hoft et al. performed the direct whole blood MGIA as part of a clinical trial of AERAS-422; 180

a live recombinant BCG expressing perfringolysin and three M.tb antigens: 85A, 85B, and 181

Rv3407[15]. Between November 2010 and August 2011, 24 volunteers were enrolled 182

(AERAS-422 high dose, n=8; AERAS-422 low dose, n=8; Tice BCG, n=8). High dose 183

AERAS-422 vaccination induced Ag85A- and Ag85B-specific lymphoproliferative responses 184

and marked anti-mycobacterial activity detectable in the direct whole blood MGIA[15]. A 185

systems biology approach using modular analysis identified positive correlations between 186

post-vaccination T cell gene expression and MGIA activity, and negative correlations 187

between post-vaccination monocyte gene expression modules and MGIA activity. 188

Unfortunately, the development of varicella-zoster virus reactivations occurred in two of 189

eight vaccine recipients given the high dose of AERAS-422, resulting in discontinuation of 190

AERAS-422 clinical development. However, these results demonstrated that the direct whole 191

blood MGIA can detect vaccine-induced increases in anti-mycobacterial immune 192

activity[15]. 193

4) Assessing M.tb infection status 194

As described, there was a significant correlation in outcome using BCG and M.tb in the direct 195

whole blood MGIA. However, no difference was detected in the magnitude of growth 196

inhibition between healthy M.tb infected and non-infected subjects using either mycobacterial 197

strain in the TB endemic region of South Africa (Baguma et al. submitted). This was in 198

contrast to a previous study in which growth of luminescent mycobacteria was significantly 199

lower in blood from tuberculin skin test (TST) positive subjects compared with TST negative 200


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subjects from a non-endemic region[10]. The discordance between these findings may have 201

resulted from the cross-sectional designs in which infection with M.tb was defined using a 202

single IGRA or TST, and it is not known if volunteers from either study were recently or 203

historically exposed to M.tb. Furthermore, despite being QuantiFERON® negative, the South 204

African volunteers had readily detectable T cell responses to BCG stimulation, suggesting 205

high levels of immunological sensitization to mycobacteria (likely due to childhood BCG 206

vaccination and/or exposure to environmental mycobacteria). 207

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Immune mechanisms of growth inhibition 209

One major goal of this project was to elucidate immune mechanisms underlying the 210

mycobacterial growth inhibition observed. The influence of IFN-γ producing T cells, 211

antibodies, monocyte:lymphocyte (ML) ratio and haemoglobin were assessed. 212

1) IFN-γ producing T cells 213

The magnitude of mycobacterial growth inhibition following BCG vaccination in the Oxford 214

study did not correlate with the antigen-specific IFN-γ ELISpot response, and a boosted IFN-215

γ ELISpot response observed following BCG revaccination was not reflected in enhanced 216

MGIA activity[13]. In the South African volunteers, despite QuantiFERON®-TB Gold 217

positive individuals having greater levels of ESAT-6 and CFP-10 specific IFN-γ secreting T 218

cells, this did not translate into improved MGIA responses. These findings are consistent with 219

previous observations that in vitro mycobacterial growth inhibition does not correlate with 220

IFN-γ production[7, 8]. 221

2) Antibodies 222

Chen et al. demonstrated that IgG antibody responses to arabinomannan (AM), a 223

mycobacterial envelope polysaccharide, increased significantly following BCG vaccination 224

using samples from the Oxford trial[16]. Their studies suggest that these antibodies, 225


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particularly when targeting specific oligosaccharide epitopes, may play a functional role in 226

mycobacterial growth inhibition. Phagocytosis and intracellular growth inhibition were 227

significantly enhanced when BCG was opsonized with post-vaccination sera, and these 228

enhancements correlated with IgG titers to AM. Furthermore, increased phagolysosomal 229

fusion in M.tb infected macrophages was observed with post-vaccination serum but not pre-230

vaccination serum, indicating that intracellular growth reduction was FcR-mediated. 231

Interestingly, they also found that the antibody response at 4 weeks post BCG vaccination 232

correlated with mycobacterial growth inhibition in the direct PBMC MGIA[16]. A functional 233

role of antibodies to AM has since been further supported by murine immunization studies 234

with AM conjugate vaccines[17]. These data highlight the importance of assessing the 235

humoral compartment in MGIAs and comparing PBMC and whole blood based approaches 236

to better assess the potential contribution of antibody-mediated responses. 237

3) Monocyte-lymphocyte ratio 238

In blood samples from healthy volunteers, a greater proportion of monocytes to lymphocytes 239

(higher ML ratio) is associated with increased mycobacterial growth and an increased 240

probability of a Type I IFN transcriptional signature[18]. Altering the ML ratio in vitro 241

impacts the control of mycobacterial growth, with either very high or very low ML ratios 242

associated with poorer control in the direct PBMC MGIA[18]. This is consistent with the 243

observation that a profoundly altered ML ratio is associated with increased risk of developing 244

TB disease in non-HIV exposed infants, in HIV exposed uninfected infants, in HIV positive 245

adults starting antiretroviral therapy and in postpartum women[2, 19-21]. Accordingly, Hoft 246

et al. showed that anti-mycobacterial immune activity correlated positively with T-cell and 247

negatively with monocyte expression signatures in the Aeras 422 study[15]. Further studies 248

with animal models may better define the sub-populations of monocytes and lymphocytes 249

important for mycobacterial growth inhibition. 250


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4) Haemoglobin 251

Using samples from the Oxford BCG vaccination study, a positive correlation was observed 252

between mean corpuscular haemoglobin (Hb) and mycobacterial growth in the direct whole 253

blood MGIA[22]. Experimental addition of Hb or ferric iron to PBMC from both humans and 254

non-human primates (NHPs) resulted in increased mycobacterial growth; an effect reversed 255

by addition of the iron chelator deferoxamine. Furthermore, expression of Hb-related genes 256

correlated significantly with mycobacterial growth in whole blood, and to a lesser extent, in 257

PBMC[22]. This association between Hb/iron levels and mycobacterial growth may in part 258

explain differences in outcome between whole blood and PBMC MGIAs and should be taken 259

into account when using these assays. 260

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Animal MGIA Studies 262

Given that early clinical studies indicate the need for more investigations before a human 263

PBMC MGIA can be qualified, MGIA development using animal models of TB represents an 264

important ongoing strategy. In addition to the benefits of sample volume and reduced 265

heterogeneity described above, animal models have the important advantage that the MGIA 266

outcome can be correlated with protection from in vivo challenge with pathogenic M.tb. This 267

would then biologically validate the assay as capable of assessing effective anti-268

mycobacterial immunity. Where there is a need to test vaccine candidates for antigen dose, 269

adjuvant dose or antigen-adjuvant combinations, an MGIA could save time, animals and 270

funding. As the direct MGIA does not depend on any species-specific immune reagents, it 271

can be easily adapted for use across a range of animal species. 272

In an effort to improve inter-site reproducibility, the animal phase of the MGIA project 273

utilized a single shared batch of frozen mycobacteria. The BCG batch was prepared at Aeras 274

and tested for viability and reproducibility prior to distribution to project partners. 275


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Investigators also agreed that, for each experiment, the colony forming units (CFUs) of 276

mycobacterial stocks would be obtained and standard curves would be used to convert Bactec 277

MGIT time to positivity (TTP) data, presented as the log10 CFU of the growth count for ease 278

of data presentation and interpretation. 279

1) Murine model 280

An MGIA assay using infected macrophages cultured separately from mouse splenocytes has 281

previously demonstrated reproducible growth inhibition data at seven days of incubation[23]. 282

Using this MGIA, immune splenocytes from mice vaccinated with five different TB vaccines 283

limited mycobacterial growth in vitro compared to naïve controls. Importantly, the vaccine-284

induced MGIA correlated at a group level with protective immune responses induced in 285

experimentally-matched animals using a mouse model of pulmonary TB[23]. 286

More recent studies have focused on simplifying the mouse MGIA model. Marsay et al. 287

showed that the direct MGIA using splenocytes has the ability to detect mycobacterial growth 288

inhibition following BCG vaccination[24]. Furthermore, investigators from CBER/FDA used 289

the direct splenocyte MGIA to demonstrate reproducible in vitro protective responses from 290

mice immunized with either BCG or a subunit vaccine, and showed that these responses 291

correlated on a group level with in vivo protection data from experimentally-matched 292

mice[25]. They also confirmed that mycobacterial quantification using the Bactec MGIT 293

system correlates strongly with plating of organ homogenates and counting CFU, and is faster 294

and easier to perform than the traditional CFU method[26]. 295

BCG gives a robust and reproducible protective effect against challenge with M.tb in 296

C57BL/6 mice and this model has therefore been used for further optimization of the mouse 297

direct MGIA. Zelmer et al. have sought to enhance the sensitivity of the direct splenocyte 298

MGIA, and found that detection of vaccine-induced inhibition could be improved by 299

lowering the MOI[27]. It was also shown that the capacity to detect mycobacterial growth 300


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inhibition in BCG vaccinated mice is time-sensitive, with growth inhibition detected only at 301

the peak of the BCG immune response (approximately 6 weeks in C57BL/6 mice)[14]. As 302

there is no amplification of the antigen specific immune response in the direct MGIA, it is 303

only the ability of the cells resident in the spleen at the time of harvest that can be assessed 304

for anti-mycobacterial capacity[14]. Since BCG is a live replicating mycobacterium, there 305

can be variation in the development of the peak of the immune response and variation in the 306

persistence of BCG vaccine in lungs and spleen. This can impact the ability to reproducibly 307

select the optimum time point for measuring a vaccine response[28, 29]. However, selecting 308

the time point of the peak immune response may be less of a challenge when assessing a 309

subunit vaccine and the ability of MGIAs to assess the effectiveness of subunit vaccines has 310

shown early promise[23]. 311

2) Guinea pig model 312

Investigators at Colorado State University and Public Health England are in the process of 313

adapting the direct MGIA for use with the guinea pig model. Early indications are that 314

PBMCs provide more robust information than whole blood, and further studies with vaccine 315

candidates are in progress to examine the utility of PBMC MGIAs in the guinea pig model. 316

3) NHP model 317

The direct whole blood MGIA has also been adapted for use with non-human primate (NHP) 318

samples at Oxford, in collaboration with Sharpe et al. at Public Health England. In a study of 319

7 Rhesus macaques the assay demonstrated a strong enhancement of mycobacterial growth 320

inhibition following BCG vaccination, which was still significant following correction for 321

changes in haemoglobin[22]. Work is currently ongoing to optimize an NHP direct PBMC 322

MGIA and correlate the outcome with protection from in vivo BCG and M.tb challenge on an 323

individual animal basis. Reproduction of the MGIA in NHPs could help to inform the human 324

studies and provide biological validation of the assay. 325


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Conclusions 326

The MGIA represents a functional assay for assessing TB vaccine activity that has the 327

potential to correlate with vaccine-induced immune protection. To date, the direct whole 328

blood MGIA has demonstrated the most consistent effect across a range of different clinical 329

vaccine studies in humans. However, because this assay must be performed within hours of 330

blood draw it requires laboratory infrastructure in close proximity to the clinical site, making 331

it difficult to assess in many vaccine trials, particularly in TB endemic countries. 332

Cryopreserved PBMCs are thus the preferred specimen for logistical reasons, and the direct 333

PBMC MGIA is currently undergoing further optimization and harmonization across 334

laboratories as part of the EURIPRED collaborative infrastructure program (H. McShane and 335

R. Tanner, personal communication). Use of MGIAs in animal models of TB provides an 336

opportunity for biological validation against protection from pathogenic in vivo challenge, 337

and could be more time- and cost-effective. Animal MGIAs could also reduce the number of 338

virulent M.tb challenge experiments required to identify promising vaccine candidates for 339

progression to clinical trials[30]. 340

The results presented indicate that the MGIA has promise as a functional assay for the 341

assessment of candidate TB vaccines. It also provides a tractable model for investigating the 342

mechanisms involved in anti-mycobacterial immunity. For example, findings from this 343

project have highlighted the importance of both lymphoid and myeloid cell mediated as well 344

as antibody-mediated immunity in these assays. In the future, it would be interesting to 345

consider how exposure of an individual to other mycobacterial species or infections (such as 346

helminths) affects ability to control mycobacterial growth in vitro, and work to this end is 347

underway. The influence of regulatory immune mechanisms and suppressors, which will be 348

represented in unbiased samples such as whole blood and PBMC, should also be explored. 349


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In this report, we have summarized the numerous accomplishments of this project. Many 350

parameters of a standardized MGIA that can be routinely performed in a laboratory with 351

access to BACTEC MGIT equipment have been established. However, MGIAs are 352

technically demanding and it is clear that further work is required, particularly to reduce the 353

variability intrinsic to functional assays. Use of cryopreserved PBMCs remains a challenge, 354

as does the goal of qualifying this assay for use in human studies in particular. Utilization of 355

the direct MGIA in animal models (especially mice and NHPs) for assessing new TB 356

vaccines seems more valuable at this time. A number of explorations into MGIAs as a 357

functional TB test should be performed in the future, and some of these goals are outlined in 358

Table 2. If validated and shown to reproducibly and consistently correlate with efficacy 359

demonstrated in clinical trials of candidate vaccines for TB, MGIAs hold great potential. 360

Much as neutralizing antibodies are currently used to assess the efficacy of certain other 361

vaccines, MGIAs could be applied for regulatory purposes as a surrogate marker of efficacy. 362

363

Acknowledgments 364

We would like to thank Robert S. Wallis for serving as a consultant on the MGIA project. We 365

thank Megan Fitzgerald, Veerabadran Dheenadhalayan and Nathalie Cadieux of Aeras for 366

production and distribution of the BCG standard for the MGIA. We also thank Lewis 367

Schrager and Barry Walker for critical comments on the manuscript. We are indebted to 368

Thomas Evans, former CEO of Aeras, for his support of the MGIA project. I thank Pere-Joan 369

Cardona for the portrait (MJB). 370

371

Ethics Statement 372

Participants in the BCG vaccination and revaccination study were recruited under a protocol 373

approved by the Oxfordshire Research Ethics Committee (OxREC A). Written informed 374


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consent was obtained from all individuals prior to enrollment in the trial. Human studies 375

performed at the South African Tuberculosis Vaccine Initiative were approved by the Human 376

Research Ethics Committee of the University of Cape Town. All adult participants provided 377

written informed consent prior to participation in the studies. In the case of children, written 378

informed consent was provided by a parent or legal guardian. All of the work at Saint Louis 379

University was approved by the Saint Louis University IRB, and PBMC from independent 380

healthy BCG vaccinated subjects were collected under a protocol approved by the 381

Institutional Review Board of the Albert Einstein College of Medicine. All animal studies 382

were approved by the appropriate institution which used IACUC-approved protocols and 383

methods. At Public Health England, all animal procedures and study design were approved 384

by the Public Health England, Porton Down Ethical Review Committee, and authorized 385

under an appropriate UK Home Office project license. 386

387

Financial Disclosure: We thank the USFDA (FDA Grant #IU18FD004012-01) and Aeras 388

for financial support. The research leading to some of these results has also received funding 389

from the European Union’s Seventh Framework Program [FP7/2007-2013] under Grant 390

Agreement No. 312661: European Research Infrastructures for Poverty Related Diseases 391

(EURIPRED); the European Union’s Horizon2020 research and innovation program under 392

Grant Agreement No. 643381; the Universities Federation for Animal Welfare (UFAW); 393

Wellcome Trust; and the Bill and Melinda Gates Foundation. 394

395

Author Contributions: Conceived and designed the experiments: MJB, HAF, RT, SM, TJS, 396

DH, HM, JMA, AI, SS, AR and DFH. Performed the experiments: RT, TJS, DH, DFH, JMA, 397

AI, SS, AR, APN, ME and AZ. Wrote the paper: MJB, HAF, RT, SM, TJS, DH, HM, JMA, 398

AI and DFH. 399


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400

Competing Interests: The authors have declared that there are no competing interests. 401

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References 403

[1] Fletcher, H. A., Snowden, M. A., Landry, B., Rida, W., Satti, I., Harris, S. A., Matsumiya, M., Tanner, 404 R., O'Shea, M. K., Dheenadhayalan, V., Bogardus, L., Stockdale, L., Marsay, L., Chomka, A., 405 Harrington-Kandt, R., Manjaly-Thomas, Z. R., Naranbhai, V., Stylianou, E., Darboe, F., Penn-406 Nicholson, A., Nemes, E., Hatheril, M., Hussey, G., Mahomed, H., Tameris, M., McClain, J. B., Evans, 407 T. G., Hanekom, W. A., Scriba, T. J., and McShane, H., 2016, "T-cell activation is an immune correlate 408 of risk in BCG vaccinated infants," Nat Commun, 7, p. 11290. 409 [2] Fletcher, H. A., Filali-Mouhim, A., Nemes, E., Hawkridge, A., Keyser, A., Njikan, S., Hatherill, M., 410 Scriba, T. J., Abel, B., Kagina, B. M., Veldsman, A., Agudelo, N. M., Kaplan, G., Hussey, G. D., Sekaly, R. 411 P., Hanekom, W. A., and team, B. s., 2016, "Human newborn bacille Calmette-Guérin vaccination and 412 risk of tuberculosis disease: a case-control study," BMC Med, 14, p. 76. 413 [3] Hanekom, W. A., Dockrell, H. M., Ottenhoff, T. H., Doherty, T. M., Fletcher, H., McShane, H., 414 Weichold, F. F., Hoft, D. F., Parida, S. K., and Fruth, U. J., 2008, "Immunological outcomes of new 415 tuberculosis vaccine trials: WHO panel recommendations," PLoS Med, 5(7), p. e145. 416 [4] Tanner, R., O'Shea, M. K., Fletcher, H. A., and McShane, H., 2016, "In vitro mycobacterial growth 417 inhibition assays: A tool for the assessment of protective immunity and evaluation of tuberculosis 418 vaccine efficacy," Vaccine. 419 [5] Wallis, R. S., Palaci, M., Vinhas, S., Hise, A. G., Ribeiro, F. C., Landen, K., Cheon, S. H., Song, H. Y., 420 Phillips, M., Dietze, R., and Ellner, J. J., 2001, "A whole blood bactericidal assay for tuberculosis," J 421 Infect Dis, 183(8), pp. 1300-1303. 422 [6] Worku, S., and Hoft, D. F., 2000, "In vitro measurement of protective mycobacterial immunity: 423 antigen-specific expansion of T cells capable of inhibiting intracellular growth of bacille Calmette-424 Guérin," Clin Infect Dis, 30 Suppl 3, pp. S257-261. 425 [7] Hoft, D. F., Worku, S., Kampmann, B., Whalen, C. C., Ellner, J. J., Hirsch, C. S., Brown, R. B., Larkin, 426 R., Li, Q., Yun, H., and Silver, R. F., 2002, "Investigation of the relationships between immune-427 mediated inhibition of mycobacterial growth and other potential surrogate markers of protective 428 Mycobacterium tuberculosis immunity," J Infect Dis, 186(10), pp. 1448-1457. 429 [8] Kampmann, B., Tena, G. N., Mzazi, S., Eley, B., Young, D. B., and Levin, M., 2004, "Novel human in 430 vitro system for evaluating antimycobacterial vaccines," Infect Immun, 72(11), pp. 6401-6407. 431 [9] Silver, R. F., Li, Q., Boom, W. H., and Ellner, J. J., 1998, "Lymphocyte-dependent inhibition of 432 growth of virulent Mycobacterium tuberculosis H37Rv within human monocytes: requirement for 433 CD4+ T cells in purified protein derivative-positive, but not in purified protein derivative-negative 434 subjects," J Immunol, 160(5), pp. 2408-2417. 435 [10] Kampmann, B., Gaora, P. O., Snewin, V. A., Gares, M. P., Young, D. B., and Levin, M., 2000, 436 "Evaluation of human antimycobacterial immunity using recombinant reporter mycobacteria," J 437 Infect Dis, 182(3), pp. 895-901. 438 [11] Cheon, S. H., Kampmann, B., Hise, A. G., Phillips, M., Song, H. Y., Landen, K., Li, Q., Larkin, R., 439 Ellner, J. J., Silver, R. F., Hoft, D. F., and Wallis, R. S., 2002, "Bactericidal activity in whole blood as a 440 potential surrogate marker of immunity after vaccination against tuberculosis," Clin Diagn Lab 441 Immunol, 9(4), pp. 901-907. 442 [12] Kolibab, K., Parra, M., Yang, A. L., Perera, L. P., Derrick, S. C., and Morris, S. L., 2009, "A practical 443 in vitro growth inhibition assay for the evaluation of TB vaccines," Vaccine, 28(2), pp. 317-322. 444 [13] Fletcher, H. A., Tanner, R., Wallis, R. S., Meyer, J., Manjaly, Z. R., Harris, S., Satti, I., Silver, R. F., 445 Hoft, D., Kampmann, B., Walker, K. B., Dockrell, H. M., Fruth, U., Barker, L., Brennan, M. J., and 446


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McShane, H., 2013, "Inhibition of mycobacterial growth in vitro following primary but not secondary 447 vaccination with Mycobacterium bovis BCG," Clin Vaccine Immunol, 20(11), pp. 1683-1689. 448 [14] Zelmer, A., Tanner, R., Stylianou, E., Damelang, T., Morris, S., Izzo, A., Williams, A., Sharpe, S., 449 Pepponi, I., Walker, B., Hokey, D. A., McShane, H., Brennan, M., and Fletcher, H., 2016, "A new tool 450 for tuberculosis vaccine screening: Ex vivo Mycobacterial Growth Inhibition Assay indicates BCG-451 mediated protection in a murine model of tuberculosis," BMC Infect Dis, 16, p. 412. 452 [15] Hoft, D. F., Blazevic, A., Selimovic, A., Turan, A., Tennant, J., Abate, G., Fulkerson, J., Zak, D. E., 453 Walker, R., McClain, B., Sadoff, J., Scott, J., Shepherd, B., Ishmukhamedov, J., Hokey, D. A., 454 Dheenadhayalan, V., Shankar, S., Amon, L., Navarro, G., Podyminogin, R., Aderem, A., Barker, L., 455 Brennan, M., Wallis, R. S., Gershon, A. A., Gershon, M. D., and Steinberg, S., 2016, "Safety and 456 Immunogenicity of the Recombinant BCG Vaccine AERAS-422 in Healthy BCG-naïve Adults: A 457 Randomized, Active-controlled, First-in-human Phase 1 Trial," EBioMedicine, 7, pp. 278-286. 458 [16] Chen, T., Blanc, C., Eder, A. Z., Prados-Rosales, R., Souza, A. C., Kim, R. S., Glatman-Freedman, A., 459 Joe, M., Bai, Y., Lowary, T. L., Tanner, R., Brennan, M. J., Fletcher, H. A., McShane, H., Casadevall, A., 460 and Achkar, J. M., 2016, "Association of Human Antibodies to Arabinomannan With Enhanced 461 Mycobacterial Opsonophagocytosis and Intracellular Growth Reduction," J Infect Dis. 462 [17] Prados-Rosales, R., Carreño, L., Cheng, T., Blanc, C., Weinrick, B., Malek, A., Lowary, T. L., Baena, 463 A., Joe, M., Bai, Y., Kalscheuer, R., Batista-Gonzalez, A., Saavedra, N. A., Sampedro, L., Tomás, J., 464 Anguita, J., Hung, S. C., Tripathi, A., Xu, J., Glatman-Freedman, A., Jacobs, W. R., Chan, J., Porcelli, S. 465 A., Achkar, J. M., and Casadevall, A., 2017, "Enhanced control of Mycobacterium tuberculosis 466 extrapulmonary dissemination in mice by an arabinomannan-protein conjugate vaccine," PLoS 467 Pathog, 13(3), p. e1006250. 468 [18] Naranbhai, V., Fletcher, H. A., Tanner, R., O'Shea, M. K., McShane, H., Fairfax, B. P., Knight, J. C., 469 and Hill, A. V., 2015, "Distinct Transcriptional and Anti-Mycobacterial Profiles of Peripheral Blood 470 Monocytes Dependent on the Ratio of Monocytes: Lymphocytes," EBioMedicine, 2(11), pp. 1619-471 1626. 472 [19] Naranbhai, V., Moodley, D., Chipato, T., Stranix-Chibanda, L., Nakabaiito, C., Kamateeka, M., 473 Musoke, P., Manji, K., George, K., Emel, L. M., Richardson, P., Andrew, P., Fowler, M., Fletcher, H., 474 McShane, H., Coovadia, H. M., Hill, A. V., and Team, H. P., 2014, "The association between the ratio 475 of monocytes: lymphocytes and risk of tuberculosis among HIV-infected postpartum women," J 476 Acquir Immune Defic Syndr, 67(5), pp. 573-575. 477 [20] Naranbhai, V., Kim, S., Fletcher, H., Cotton, M. F., Violari, A., Mitchell, C., Nachman, S., 478 McSherry, G., McShane, H., Hill, A. V., and Madhi, S. A., 2014, "The association between the ratio of 479 monocytes:lymphocytes at age 3 months and risk of tuberculosis (TB) in the first two years of life," 480 BMC Med, 12, p. 120. 481 [21] Naranbhai, V., Hill, A. V., Abdool Karim, S. S., Naidoo, K., Abdool Karim, Q., Warimwe, G. M., 482 McShane, H., and Fletcher, H., 2014, "Ratio of monocytes to lymphocytes in peripheral blood 483 identifies adults at risk of incident tuberculosis among HIV-infected adults initiating antiretroviral 484 therapy," J Infect Dis, 209(4), pp. 500-509. 485 [22] Tanner, R., O'Shea, M. K., White, A. D., Müller, J., Harrington-Kandt, R., Matsumiya, M., Dennis, 486 M. J., Parizotto, E. A., Harris, S., Stylianou, E., Naranbhai, V., Bettencourt, P., Drakesmith, H., Sharpe, 487 S., Fletcher, H. A., and McShane, H., 2017, "The influence of haemoglobin and iron on in vitro 488 mycobacterial growth inhibition assays," Sci Rep, 7, p. 43478. 489 [23] Parra, M., Yang, A. L., Lim, J., Kolibab, K., Derrick, S., Cadieux, N., Perera, L. P., Jacobs, W. R., 490 Brennan, M., and Morris, S. L., 2009, "Development of a murine mycobacterial growth inhibition 491 assay for evaluating vaccines against Mycobacterium tuberculosis," Clin Vaccine Immunol, 16(7), pp. 492 1025-1032. 493 [24] Marsay, L., Matsumiya, M., Tanner, R., Poyntz, H., Griffiths, K. L., Stylianou, E., Marsh, P. D., 494 Williams, A., Sharpe, S., Fletcher, H., and McShane, H., 2013, "Mycobacterial growth inhibition in 495 murine splenocytes as a surrogate for protection against Mycobacterium tuberculosis (M. tb)," 496 Tuberculosis (Edinb), 93(5), pp. 551-557. 497


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[25] Yang, A. L., Schmidt, T. E., Stibitz, S., Derrick, S. C., Morris, S. L., and Parra, M., 2016, "A 498 simplified mycobacterial growth inhibition assay (MGIA) using direct infection of mouse splenocytes 499 and the MGIT system," J Microbiol Methods, 131, pp. 7-9. 500 [26] Kolibab, K., Yang, A., Parra, M., Derrick, S. C., and Morris, S. L., 2014, "Time to detection of 501 Mycobacterium tuberculosis using the MGIT 320 system correlates with colony counting in 502 preclinical testing of new vaccines," Clin Vaccine Immunol, 21(3), pp. 453-455. 503 [27] Zelmer, A., Tanner, R., Stylianou, E., Morris, S., Izzo, A., Williams, A., Sharpe, S., Pepponi, I., 504 Walker, B., Hokey, D., McShane, H., Brennan, M., and Fletcher, H., 2015, "Ex vivo mycobacterial 505 growth inhibition assay (MGIA) for tuberculosis vaccine testing - a protocol for mouse 506 splenocytes,"BioRxiv. 507 [28] Kaveh, D. A., Carmen Garcia-Pelayo, M., and Hogarth, P. J., 2014, "Persistent BCG bacilli 508 perpetuate CD4 T effector memory and optimal protection against tuberculosis," Vaccine, 32(51), 509 pp. 6911-6918. 510 [29] Olsen, A. W., Brandt, L., Agger, E. M., van Pinxteren, L. A., and Andersen, P., 2004, "The 511 influence of remaining live BCG organisms in vaccinated mice on the maintenance of immunity to 512 tuberculosis," Scand J Immunol, 60(3), pp. 273-277. 513 [30] Tanner, R., and McShane, H., 2017, "Replacing, reducing and refining the use of animals in 514 tuberculosis vaccine research," ALTEX, 34(1), pp. 157-166. 515 516

517

Figure Legends 518

519

Figure 1: Schematic of the MGIT mycobacterial growth inhibition assay method. Whole 520

blood or cell culture medium containing the appropriate concentration of PBMC (or mouse 521

splenocytes in murine studies) is inoculated with an equal volume of mycobacteria at a low 522

multiplicity of infection (MOI) (~1 CFU:10,000 PBMC). Cultures are incubated in 2ml tubes 523

at 37°C for 96 hours with 360° rotation. Cells are then lysed to remove red blood cells and/or 524

release intracellularised mycobacteria. The lysate is inoculated into BACTEC mycobacteria 525

indicator tubes (MGITs). Tubes are placed on the BACTEC 960 machine and incubated at 526

37°C until detection of positivity by fluorescence. On day 0, direct-to-MGIT viability 527

controls are set up by directly inoculating MGIT tubes with the same volume of mycobacteria 528

as the samples. The time to positivity (TTP) is converted to a log10 CFU value using a 529

standard curve and the final value is expressed as an absolute CFU value or relative to the 530

control, indicating the amount of growth inhibition that has occurred during the culture 531

period. 532


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Figure 2: Direct whole blood MGIA in South African infants. A) Whole blood samples 533

from 10 South African infants were assessed using the direct whole blood MGIA. There was 534

a significant correlation between growth of BCG SSI and M.tb H37Rv (Spearman’s r = 0.73, 535

p = 0.02). B) Whole blood samples from 10 South African infants were compared with those 536

from 53 South African adults using the direct whole blood MGIA. There was a significant 537

reduction in growth of BCG SSI in infants compared with adults (A Mann-Whitney U test 538

was performed between groups where *represents a p-value of <0.05). 539

540

Tables 541

542

Table 1: Major early objectives of the MGIA project 543

544

Major Early Objectives 545

546

Assess the variability, reproducibility and transferability of four different MGIAs in 547

naïve, BCG vaccinated and BCG re-vaccinated subjects. 548

549

Compare PBMC and WB MGIA responses induced by wild type BCG and a new 550

recombinant BCG vaccine. 551

552

In a TB endemic population (South Africa), determine the effect of M.tb infection 553

status on inhibition of mycobacterial growth. 554

555

Test sera obtained from subjects enrolled in the enhanced BCG vaccine MGIA study 556

for antibodies to capsular and cell surface constituents including major polysaccharide 557

antigens and to compare the pre- to post-immunization antibody levels with MGIA 558

outcomes. 559

560

Explore other immune mechanisms underlying mycobacterial growth inhibition (eg. 561

by correlating to ELISPOT and ICS-Flow analysis on blood samples from the BCG 562

study cohorts). 563

564

565


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Table 2: Future goals of MGIA development 566

567

Future goals of MGIA development 568

569

Demonstrate that the direct PBMC MGIA meets requirements as a qualified assay 570

571

Determine if there are significant differences when using BCG or M.tb strains for in 572

vitro infection 573

574

Evaluate differences when using MGIAs in endemic or non-endemic countries 575

576

Determine if MGIAs can be used in M.tb infected populations 577

578

Identify factors in PBMC and whole blood that mediate and influence growth 579

inhibition 580

581

Evaluate performance of PBMC-MGIT in trials of novel TB vaccine candidates 582

583

Determine if the test is reproducible at different sites 584

585

Determine if the direct whole blood MGIA can be further adapted or refined for use in 586

vaccine or TB studies 587

588

589

590

Biographical Text 591

592

Dr. Michael J. Brennan: I was the first one in my family to go to college. I owe a great deal 593

to my parents and sister who sacrificed much so that I could take the time needed to become a 594

scientist. To have experienced a career I love, I have been exceptionally fortunate. 595

Throughout, I have held different positions at the National Institutes of Health, at the World 596

Health Organization and I eventually retired from the U.S Food and Drug Administration. 597

Now near the end of my career, I am employed by the non-profit organization, Aeras, and 598

have learned many new things about the manufacturing and clinical investigation of vaccines, 599

especially those for Tuberculosis. All over, I have travelled to six continents, participated in 600


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many valuable conferences, and had meaningful and insightful conversations with hundreds 601

of scientists. Perhaps, those conversations have been the best part of all. 602

Dr. Rachel Tanner is a post-doctoral scientist at the Jenner Institute, University of Oxford. 603

She obtained a BA (Hons) in Biological Sciences at University of Oxford and developed an 604

early interest in vaccine immunology, taking up a Research Assistant post at the Centre for 605

HIV/AIDS Vaccine Immunology (CHAVI). Dr. Tanner received her DPhil in Clinical 606

Medicine at the Jenner Institute, University of Oxford in 2015. Her PhD project was 607

concerned with the development and optimisation of an MGIA across multiple species, and 608

she continues to work on such functional assays as part of her post-doctoral research. Dr. 609

Tanner’s broader interests are in immune correlates and the host immune response to TB 610

vaccination. 611

Dr. Sheldon Morris is a retired scientist who worked in the Laboratory of Mycobacterial 612

Diseases and Cellular Immunology at CBER/FDA from 1986-2015. He served as Laboratory 613

Chief from 2001-2015. Dr. Morris’s activities included the regulatory evaluation of 614

tuberculosis and malaria vaccines and scientific research involving the development of new 615

tuberculosis vaccines and novel assays to support vaccine development. During his tenure at 616

CBER, Dr. Morris authored or co-authored more than 100 publications on mycobacterial 617

disease research and was invited to give 52 scientific presentations. In addition, he was a 618

member of the Editorial Board for Antimicrobial Agents and Chemotherapy and served as an 619

ad hoc reviewer for 13 other journals. Importantly, Dr. Morris was selected to participate on 620

11 National Institute of Health grant review panels, 3 Welcome Trust grant reviews, and was 621

a scientific advisor to the World Health Organization during 4 meetings. 622

Dr. Thomas J. Scriba trained in Biological Sciences at Stellenbosch University, South 623

Africa and in T cell Immunology at Oxford University, United Kingdom. He returned to 624

South Africa for postdoctoral training in Paediatric and Clinical Immunology in Tuberculosis 625


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and Vaccinology at the Institute of Infectious Disease and Molecular Medicine (IDM), 626

University of Cape Town. Dr. Scriba is Associate Professor at the University of Cape Town, 627

South Africa and is Deputy Director, Immunology at the South African Tuberculosis Vaccine 628

Initiative. His research interests include clinical immunology of tuberculosis and 629

development of novel prevention strategies against tuberculosis. 630

Dr. Jacqueline M. Achkar, M.D., M.Sc., is a physician scientist and an Associate Professor 631

in the Departments of Medicine (Division of Infectious Diseases) and Microbiology & 632

Immunology at the Albert Einstein College of Medicine, Bronx, New York. She received her 633

Medical Diploma and Doctoral Degree from the Free University of Berlin, Germany, her 634

Master of Science Degree in Clinical Research Methods from the Albert Einstein College of 635

Medicine, and completed her Medical Residency and Infectious Disease Fellowship at the 636

New York University School of Medicine. Dr. Achkar leads a successful translational 637

research program in the field of tuberculosis serology and biomarker discovery which 638

includes the identification of human antibody targets and the study of antibody functions in 639

the defense against TB. 640

Dr. Andrea Zelmer received her degree in Biology in 2002 and PhD in Infection Biology in 641

2005 from the Technical University Braunschweig, Germany. She subsequently moved to 642

London, UK to start a post-doctoral position at the UCL School of Pharmacy to work on 643

treatment and pathogenesis of E. coli K1 infection in neonates. In 2008 Dr. Zelmer moved to 644

the London School of Hygiene and Tropical Medicine to develop imaging technologies and 645

mouse models for testing of new drugs and vaccines against tuberculosis. She is now an 646

Assistant Professor at LSHTM. Over the last three years Dr. Zelmer has been focusing on 647

developing clinically relevant animal models for vaccine testing and studying the immune 648

response to vaccination. 649


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Dr. David Hokey is the Senior Director of Human Immunology at Aeras in Rockville, 650

Maryland. He received his Ph.D. in Immunology from the University of Pittsburgh in 2005 651

where he worked primarily on dendritic cell-based tumor vaccines. He then performed his 652

postdoctoral research under the guidance of Dr. David Weiner at the University of 653

Pennsylvania which focused on DNA vaccines for HIV, cancer, and influenza with an 654

emphasis on primate immunology and polychromatic flow cytometry. Dr. Hokey’s current 655

work at Aeras involves evaluation of immune responses to TB vaccine candidates in pre-656

clinical and clinical settings. His research interests include evaluation of novel vaccine 657

platforms and adjuvants for manipulation of immune responses as well as assay optimization 658

and he has worked in this area for 18 years. 659

Dr. Angelo Izzo received his B.Sc. (Hons) and PhD at the University of Adelaide, South 660

Australia. He then undertook post-graduate training at the Universita di Milano, Instituto di 661

Virologia, Milan, Italy and returned to the University of Adelaide as a Research Assistant. 662

Dr. Izzo later took up a Post-doctoral fellowship at the Trudeau Institute, Saranac Lake, New 663

York followed by the University of New Mexico School of Medicine, Albuquerque, New 664

Mexico. He then became Assistant Professor at Midwestern University Department of 665

Microbiology, Illinois and is now Assistant Professor at Colorado State University, Fort 666

Collins, Colorado. Dr. Izzo has over 25 years’ experience investigating the immune response 667

to pulmonary Mycobacterium tuberculosis infection in animal models. He has obtained 668

extensive experience in understanding vaccine induced immunology and 669

immunopathogenesis of pulmonary tuberculosis using the animal models. 670

Dr. Sally Sharpe is a Scientific Leader at Public Health England, Porton Down. She received 671

a B.Sc. from London University in 1984 then took up a Research Assistant post at The Centre 672

of Applied Microbiology and Research at Porton Down where she was trained in T cell 673

immunology and flow cytometry. She worked within a team developing non-human primate 674


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models of HIV and was awarded a PhD (Open University, 1997) for research into T cell 675

responses to simian immunodeficiency virus. Since 2003, she has led work to develop refined 676

macaque models of Tuberculosis for the evaluation of new interventions at the Health 677

Protection Agency and subsequently Public Health England on the Porton Down site. 678

Dr. Ann Rawkins nee Williams obtained her B.Sc. at the University of Wales in Cardiff and 679

began her career as a Research Assistant studying bacterial pathogenesis at the Centre of 680

Applied Microbiology and Research at Porton Down in 1985. She conducted her post-681

graduate studies on the pathogenic mechanisms of Legionella pneumophila and was awarded 682

a PhD in 1995. After a brief period investigating Salmonella infection of poultry, she began 683

M. tuberculosis research, developing aerosol-challenge in-vivo models for pathogenesis 684

studies and subsequently for the evaluation of interventions, predominantly vaccines. The 685

vaccine evaluation team which she established is internationally renowned and collaborates 686

with the majority of TB vaccine developers and she has been involved in multiple EU-funded 687

consortia. She is currently a Scientific Leader providing scientific direction to the TB 688

research programme at the Public Health England laboratories at Porton Down, Salisbury, 689

UK. She has published widely in the name of Williams. 690

Dr. Adam Penn-Nicholson is a Research Officer/Scientist at the South African Tuberculosis 691

Vaccine Initiative, at University of Cape Town, South Africa. He previously worked as a 692

postdoctoral Fellow at the University of Cape Town. Dr. Penn-Nicholson received his PhD 693

at Case Western Reserve University, in Cleveland, Ohio, USA. Dr. Penn-Nicholson’s main 694

focus is the discovery of blood-based biomarkers that prospectively predict TB disease risk, 695

and understanding the biology involved in progression from latent M. tuberculosis infection 696

to active TB disease. He also provides scientific oversight of immunology for several clinical 697

TB vaccine trials currently being conducted at SATVI. 698


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Mr Mzwandile Erasmus has a BSc from the University of Cape Town and joined the South 699

African Tuberculosis Vaccine Initiative (SATVI) shortly after graduating in 2007. He is 700

currently a Senior Laboratory Technologist has been involved in multiple clinical and 701

research studies conducted at SATVI. 702

Dr. Elena Stylianou has a BSc. and MSc. in Biochemistry from Imperial College, London 703

and a PhD in Immunology from St George’s, University of London. She has since been at the 704

Jenner Institute, University of Oxford, training as a post-doctoral immunologist. Dr. 705

Stylianou has always found the complexity of the interaction between Mycobacterium 706

tuberculosis (M.tb) and the human host fascinating and both her PhD and post-doctoral 707

studies focused on pre-clinical vaccine research against tuberculosis. The key areas of her 708

research include mucosal and systemic delivery of viral vectors, immune responses after 709

vaccination and evaluation of vaccine protective efficacy using M.tb infection models. 710

Dr. Dan F. Hoft M.D., Ph.D. is the Director of the Division of Infectious Diseases, Allergy 711

& Immunology at Saint Louis University. Dr. Hoft was trained in Infectious Diseases at the 712

University of Iowa, where he received an NIH Physician Science Training Award (PSTA) 713

allowing him to complete a Ph.D. in microbiology/immunology. He came to Saint Louis 714

University in 1992 and has received continuous NIH funding since his arrival. In 2006, he 715

was named the Director of the new Division of Immunobiology. In 2010, the Divisions of 716

Immunobiology, Infectious Diseases and Allergy & Immunology were joined into the new 717

Division of Infectious Diseases, Allergy & Immunology with Dr. Hoft as Director. In 2014 718

he became the Principal Investigator of SLU’s Vaccine & Treatment Evaluation Unit 719

(VTEU). The capacity to induce protective immune memory by vaccination is a powerful 720

public health tool, and Dr. Hoft has been working in this area for 32 years. 721

Professor Helen McShane is currently a Professor of Vaccinology at Oxford University, 722

Wellcome Senior Clinical Research Fellow, Deputy Head of Department for Nuffield 723


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Department of Medicine and Honorary Consultant physician in HIV medicine. Professor 724

McShane obtained an intercalated BSc in 1988, followed by a degree in medicine in 1991 725

(both University of London). In 1997 She was awarded an MRC Clinical Training Fellowship 726

to undertake a PhD with Adrian Hill in Oxford, and was later awarded a PhD in 2001 727

(University of London). In 2001 she was awarded a Wellcome Clinician Scientist Fellowship, 728

allowing her to complete her clinical training and subsequently awarded a CCST in HIV and 729

GU Medicine in 2003. In 2005, she was awarded a Wellcome Senior Clinical Research 730

Fellowship; this Fellowship was renewed in 2010. Professor McShane has led the TB vaccine 731

research group at Oxford University for 16 years. 732

Dr. Helen A. Fletcher is an Associate Professor of Immunology and Deputy Director of the 733

TB Centre at the London School of Hygiene & Tropical Medicine (LSHTM). Dr. Fletcher 734

has worked in the field of TB immunology for 17 years at University College London, 735

University of Oxford and LSHTM. She has used transcriptomics and cellular immunology to 736

identify correlates of TB disease risk including increased monocyte to lymphocyte ratio and 737

increased CD4+ T cell activation in South African infants at risk of developing TB disease. 738

Dr. Fletcher has published more than 90 papers and her current work at LSHTM continues to 739

focus on immune correlates of risk and the host response to TB vaccination. 740


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