Recombinant BCG prime-recombinant adenovirus boost...

Revised Manuscript 1

2

Recombinant BCG prime-recombinant 3

adenovirus boost vaccination in rhesus monkeys 4

elicits robust polyfunctional 5

SIV-specific T cell responses 6

7

Mark J. Cayabyab1,5

, Birgit Korioth-Schmitz1, Yue Sun

1, Angela Carville

2, Harikrishnan 8

Balachandran1, Ayako Miura

1, Kevin R. Carlson

1, Adam P. Buzby

1, Barton F. Haynes

3, 9

William R. Jacobs4, and Norman L. Letvin

1* 10

11

Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 12

022151; New England Primate Research Center, Harvard Medical School, Southborough, 13

MA 017722; Duke University School of Medicine, Durham, North Carolina 27710

3; and 14

the Department of Microbiology and Immunology, Howard Hughes Medical Institute, 15

Albert Einstein College of Medicine, Bronx, New York 104614 16

17

5 Present address: The Forsyth Institute, 140 The Fenway, Boston, MA 02115 18

19

*Corresponding author. Mailing address: Department of Medicine, Division of Viral 20

Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Center for 21

Life Science, 330 Brookline Avenue, Boston, MA 02215. Phone: (617) 735-4400. Fax: 22

(617) 735-4527. Email: [email protected] 23

24

25

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Virol. doi:10.1128/JVI.02544-08 JVI Accepts, published online ahead of print on 18 March 2009

on May 7, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

Abstract 26

27

While mycobacteria have been proposed as vaccine vectors because of their persistence 28

and safety, little has been done systematically to optimize their immunogenicity in 29

nonhuman primates. We successfully generated rBCG expressing SIV Gag and Pol as 30

multigenic, non-integrating vectors, but rBCG expressing SIV Env was unstable. A dose 31

and route determination study in rhesus monkeys revealed that intramuscular 32

administration of rBCG was associated with local reactogenicity; whereas intravenous 33

and intradermal administration of 106 to 10

8 cfu rBCG was well-tolerated. Following 34

single or repeat rBCG inoculations, monkeys developed high frequency BCG PPD IFNγ 35

ELISpot responses. However, those same animals developed only modest SIV-specific 36

CD8+ T cell responses. Nevertheless, high frequency SIV-specific cellular responses 37

were observed in the rBCG-primed monkeys following boosting with rAd5 expressing 38

the SIV antigens. These cellular responses were of greater magnitude and more persistent 39

than those generated after vaccination with rAd5 alone. The vaccine-elicited cellular 40

responses were predominantly polyfunctional CD8+ T cells. These findings support the 41

further exploration of mycobacteria as priming vaccine vectors. 42

43

44


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

Introduction 45

46

Because of its proven safety and immunogenicity, Mycobacterium bovis Bacillus 47

Calmette Guérin (BCG) is currently being explored as a vaccine vector to confer 48

protection against infectious pathogens and cancer. Recombinant BCG (rBCG) has been 49

constructed to express a variety of antigens, including proteins of HIV-1, tuberculosis, 50

and parasites (7, 12, 18, 24, 29, 38). However, rBCG has not demonstrated consistent 51

protection and has failed in human clinical trials as a Lyme disease vaccine (11). While 52

the disappointing protection seen in these studies may be a consequence of transgene 53

expression instability or inefficient antigen processing and presentation (9), rBCG 54

constructs may simply not be sufficiently immunogenic to elicit protective immunity 55

when administered intradermally as stand-alone vaccines. 56

Heterologous prime/boost vaccination regimens employing recombinant bacteria, 57

viruses and protein have been shown to generate more robust cellular immune responses 58

than vaccine strategies employing a simple vaccine modality. In efforts to improve BCG 59

as a vaccine against tuberculosis, prime/boost strategies using BCG as prime and 60

heterologous constructs such as recombinant adenovirus 5 (rAd5), DNA and recombinant 61

poxviruses as boosting immunogens were shown to enhance CD4+ and CD8

+ T cell 62

responses to BCG antigens (14, 28, 33, 36, 43-45). A heterologous prime/boost 63

immunization approach has also been shown to increase the immunogenicity of HIV-64

1/SIV antigens expressed by recombinant BCG and other mycobacteria vectors. Ami et 65

al. have shown that priming with rBCG expressing SIV Gag and boosting with a 66

recombinant vaccinia virus generated higher virus-specific cellular responses in 67

vaccinated monkeys than either rBCG or recombinant vaccinia virus alone (2). We 68

recently showed that a recombinant mycobacteria M. smegmatis vector expressing an 69


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

HIV-1 envelope protein was able to generate virus-specific CD8+ T cell memory 70

responses in mice, and boosting with rAd5 substantially increased CD8+ T cell responses 71

specific for that HIV-1 antigen (6, 15). 72

In the current study, we assessed the immunogenicity of rBCG expressing SIV 73

Gag, Pol and Env antigens in rhesus monkeys using escalating doses of rBCG and 74

various routes of administration. We also explored whether we can augment the immune 75

responses elicited by rBCG by boosting with a replication-defective rAd5 vector. We 76

demonstrate that intradermal and intravenous administration of rBCG are better tolerated 77

than other routes of rBCG administration and that rBCG prime/rAd5 vector boost 78

vaccination induced robust, persistent and polyfunctional CD8+ T cell responses. 79

80

81


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

Material and Methods 82

83

Generation of recombinant BCG. Mycobacterium bovis BCG (Pasteur) was grown in 84

7H9 media supplemented with 10% oleic acid-albumin-dextrose-catalase (Difco) and 85

0.05% Tween 80 (Fisher Scientific). Full-length SIVmac239 gag and pol and a modified 86

env were cloned from codon-optimized DNA plasmids constructed previously (20, 23). 87

The SIV env was modified by removing the signal sequence and the gp41 region. The 88

SIV genes were then individually inserted into the multicopy pJH222 Escherichia 89

coli/mycobacteria shuttle plasmid (6). A synthetic operon was constructed containing

the 90

viral genes, regulated by the M. tuberculosis α antigen promoter and the M. tuberculosis 91

19-kDa signal sequence. For detection of the SIV proteins, a hemagglutinin

(HA) tag was 92

fused to the C-terminal end of each viral protein. Within the operon, a kanamycin 93

resistance gene was cloned downstream of the viral gene. Each individual multicopy 94

plasmid with the SIV gag, pol or env gene insert was transformed into BCG (Pasteur). 95

Recombinant mycobacterial clones were selected for kanamycin resistance on 7H10 agar 96

containing 20 µg/ml kanamycin (Sigma). Single colonies were grown

in 7H9 medium 97

containing 20 µg/ml of kanamycin and grown by shaking until they reached an optical 98

density at 600 nm (OD600) approximately equal to 1. Mycobacteria were then harvested

99

and washed twice in ice-cold phosphate-buffered saline (PBS). Expression of the SIV 100

proteins was assessed by Western blotting of mycobacterial lysates (1 µg of total protein)

101

using an anti-HA monoclonal antibody (MAb) (clone 3F10) and a chemiluminescence 102

detection kit according to the manufacturer's protocol (Roche Applied Science). 103

104

Animals and Immunization. The Mamu-A*01+

rhesus monkeys (Macaca mulatta) used 105

in these studies were obtained from the Caribbean Primate Research Center, Puerto Rico 106


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

and housed at the New England Primate Research Center, Southborough, MA. These 107

monkeys were not specific pathogen free. They were tested to be negative for M. 108

tuberculosis infection four weeks before the study was initiated. All animals were 109

maintained in accordance with the NIH Guide to the Care and Use of Laboratory 110

Animals (30) and with the approval of the Institutional Animal Care and Use Committee 111

of Harvard Medical School and the National Institutes of Health. 112

The immunization schedule for each experimental group of monkeys is 113

summarized in Fig. 1. Monkeys were inoculated intradermally, intramuscularly or 114

intravenously with 106, 10

7, 10

8, or 10

9 colony forming units (cfu) of rBCG SIV Gag, Pol 115

and Env at weeks 0 and 23. At week 43, monkeys were boosted with recombinant 116

replication defective E1 deleted-adenovirus 5 vector expressing SIVmac239 Gag, Pol, 117

and Env (21, 25). 1010

particles of rAd5 encoding the SIV Gag-Pol fusion protein and 118

1010

particles rAd5 SIV encoding Env were administered by intramuscular injection. 119

120

IFNγ ELISPOT assays. 96-well microtiter plates (Multiscreen-Immobilon-P, 121

Millipore, Bedford, MA) were coated overnight with 5 µg/ml anti-human interferon-γ 122

(BD PharMingen), washed, and then blocked with RPMI containing 10% FBS. PBMCs 123

were counted using a Guava Easy Cyte (Guava Technologies, Hayward, CA) and 124

resuspended in 10% FBS/RPMI at a density of 2x105 cells per well in triplicates in the 125

presence of 2 µg/ml SIV antigen pooled peptides or 2 µg/ml p11C (CTPYDINQM) 126

peptide (NEP, Gardner, MA), 25 µg/well bovine PPD (Tuberculin OT, Synbiotics), 127

medium as negative control or the mitogen phytohaemagglutinin-M (PHAM; Sigma) as 128

positive control. SIV Gag, Pol, and SIV Env pooled peptides (15-mers overlapping by 11 129

amino acids) were obtained through the AIDS Research and Reference Reagent Program, 130


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

Division of AIDS, NIAID, NIH. Plates were incubated overnight at 37ºC, 5% CO2 and 131

then washed and incubated with biotinylated rabbit polyclonal anti-human interferon-γ 132

(Biosource, Camarillo, CA) for two hours at room temperature, followed by streptavidin-133

alkaline phosphatase (Southern Biotechnology, Birmingham, AL) for two hours. Plates 134

were developed using a chromogenic substrate NBT/BCIP (Pierce, Rockford, IL), and 135

read on an ImmunoSpot analyzer (Cellular Technology, Cleveland, OH) using CTL 136

ImmunoSpot image-processing software. The median IFNγ spot-forming cells (SFC) per 137

106 PBMC were calculated by subtracting the negative control from the experimental 138

median SFC counts per triplicate wells. Median SFC values that exceeded twice the 139

background were considered positive. 140

141

Tetramer staining and lymphocyte analysis. The PE-coupled tetrameric Mamu-142

A*01/p11C, C-M complex (CTPYDINQM) was used to stain CD8+

T lymphocytes 143

specific for the SIV Gag epitope p11C (17). The tetrameric complex was added at a 144

concentration of 1 µg/ml to 100 µl of whole blood for 15 minutes at room temperature or 145

to 5x105 lymphocytes, isolated by density-gradient centrifugation over Ficoll Paque after 146

10-13 days of in vitro culture with the supplementation of 20 U/ml recombinant human 147

interleukin-2 (Il-2; Hoffmann-La Roche, Nutley, NJ) after three days of culture. An 148

antibody cocktail of fluorescein isothiocyanate (FITC)-labeled anti-human CD8α (Leu2a;

149

Becton Dickinson), phycoerythrin (PE)-labeled anti-human CD4 (19Thy5D7; custom 150

conjugated by Beckman Coulter, Fullerton, CA), and allophycocyanin (APC)-labeled 151

anti-rhesus CD3 (FN18; custom conjugated by Becton Dickinson) was added for another 152

15 minutes. After whole blood staining, red blood cells were lysed in an Immunoprep 153

Reagent TQ-Prep Workstation (Beckman-Coulter). Washed cells were fixed in 1% 154


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

formaldehyde. Samples were analyzed on a FACSCalibur (BD PharMingen, San Jose, 155

CA) utilizing CellQuest software. Gated CD3+CD8α

+ T cells were examined for staining 156

with the tetrameric Mamu-A*01/p11C complex. 157

Peripheral blood CD4+ and CD8

+ T lymphocyte counts were determined by 158

multiplying the total lymphocyte count by the percentage of CD4+3

+ and CD8

+3

+ T cells 159

as assessed by flow cytometry. Complete blood counts were performed on an Advia 120 160

(Bayer Diagnostics, Tarrytown, NY). 161

162

Intracellular cytokine staining analysis. PBMCs were isolated from EDTA-163

anticoagulated blood and frozen in the vapor phase of liquid nitrogen. Cells were later 164

thawed and allowed to rest for 6 h at 37°C in a 5% CO2 environment. The viability of 165

these cells was >90%. PBMCs were then incubated at 37°C in a 5% CO2 environment 166

for 6 h in the presence of RPMI/10% fetal calf serum alone (unstimulated), a pool of 15-167

mer Gag peptides (2 µg/ml of each peptide; AIDS Research and Reference Reagent 168

Program, Division of AIDS, NIAID, NIH, Germantown, MD), or staphylococcal 169

enterotoxin B (SEB) (5 µg/ml, Sigma-Aldrich, St. Louis, MO) as a positive control. All 170

cultures contained Monensin (GolgiStop; BD Biosciences) as well as 1

µg/ml anti-CD49d 171

(9F10; BD Biosciences). The cultured cells were cell-surface stained with the following 172

mAbs: anti-CD3-Pacific Blue (SP34-2; BD Biosciences), anti-CD4-Quantum-Dot 605 173

(19Thy 5D7 – in house production, Quantum-Dot 605 was obtained from Invitrogen, 174

Carlsbad, CA), anti-CD8α-Alexa Fluor 700 (RPA-T8; BD Biosciences), and anti-CD28-175

PerCP-Cy5.5 (L293; BD Biosciences). After fixing with Cytofix/Cytoperm solution (BD 176

Biosciences), cells were permeabilized and stained with anti-IFNγ-PE-Cy7 (B27; BD 177

Biosciences), anti-TNFα-FITC (MAb11; BD Biosciences, San Jose, CA), and anti-IL-2-178


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

APC (MQ1-17H12; BD Biosciences). A violet fluorescent reactive dye (Live/Dead 179

Fixable Violet Dead Cell Stain Kit; Invitrogen) was used as a viability marker to exclude 180

dead cells. Labeled cells were fixed in 1% formaldehyde-PBS. 181

Samples were collected on a BD LSRII flow cytometer (BD Biosciences) and 182

analyzed using FlowJo software (Tree Star, Ashland, OR). Approximately 500,000 to 183

1,000,000 events were collected per sample. Doublets were excluded by forward scatter 184

(FSC)-area versus FSC-height. Dead cells were excluded by their staining with amine 185

reactive dye. CD4+ and CD8

+ T cells were determined by their expression of CD3, CD4 186

or CD8. Functional analysis was done by plotting the expression of each cytokine 187

molecule against another, and a boolean combination of single functional gates was 188

generated using FlowJo software. The frequency of cells producing IFNγ, TNFα, and IL-189

2, either individually or in any combination, was determined from the FlowJo, formatted 190

in PESTLE, and analyzed using SPICE software (both PESTLE and SPICE software 191

were provided by M. Roederer, Bethesda, NIH). All values used for analysis are 192

background subtracted. Responses were considered positive when the percentage of total 193

cytokine-producing cells was at least twice that of the background, and the cutoff for a 194

positive response was 0.05%. 195

196

Statistical analysis. Statistical analyses and graphical presentations were computed with 197

GraphPad Prism (GraphPad Software, San Diego, CA). The two-sided nonparametric 198

exact Mann-Whitney test was used to compare the cellular immune responses between 199

groups of experimental animals. Multiple groups of animals were compared using the 200

nonparametric one-way ANOVA Kruskall-Wallis test with a Dunn’s post test. A p value 201


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

of <0.05 was considered significant. Comparison of cytokine distributions between 202

groups was performed using a one-sided permutation test in SPICE. 203

204

205


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

Results 206

207

Generation of recombinant BCG expressing SIV Gag, Pol and Env. The high 208

expressor multi-copy pJH222 plasmid (6) was used to generate recombinant BCG 209

expressing the SIVmac239 Gag, Pol, and Env proteins fused to the 19-kDa signal 210

peptide. Transformation of BCG with the pJH222 plasmid containing either the gag or 211

pol gene produced clones that expressed high levels of the viral proteins. All rBCG 212

clones tested expressed the full length SIV Gag protein, while both full-length and 213

truncated forms of SIV Pol were expressed in the mycobacteria (Fig. 2). PCR analysis 214

indicated that several transformants contained truncated pol gene inserts (data not shown) 215

consistent with lower molecular weight products seen in Western blots. We had difficulty 216

expressing the full-length SIV Env gp160 (our unpublished observation). Removal of the 217

leader sequence and gp41 resulted in expression of SIV Env in BCG, albeit at low levels 218

(Fig. 2). However, pJH222 containing this modified SIV env was inefficient in 219

transforming BCG. 220

221

Route of administration and dose titration in rhesus monkeys of recombinant BCG 222

expressing SIV Gag, Pol and Env. Previous studies in small laboratory animals 223

suggested that the dose and route of administration of recombinant BCG are both 224

important for the optimal induction of immune responses (16, 34). Rhesus monkeys were 225

therefore inoculated intradermally, intravenously or intramuscularly with increasing 226

doses (106 to 10

9 cfu) of rBCG expressing SIV Gag, Pol and Env. We observed severe 227

reactogenicity at the inoculation site in monkeys injected intramuscularly with rBCG. 228

Therefore, this route of rBCG administration was not considered in evaluating the 229

immunogenicity of these vaccine constructs. For evaluating the immunogenicity of these 230


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

constructs, Mamu-A*01+ rhesus monkeys were inoculated with rBCG either 231

intradermally or intravenously at escalating doses (Fig. 1). Two monkeys received 106, 232

107, 10

8 and 10

9 cfu injected intradermally at four different sites on the back. At doses of 233

108 and 10

9 cfu bacilli injected intradermally, the rBCG vaccine constructs were also 234

reactogenic in the monkeys. However, the lesions resolved in 3 to 4 weeks. Eight Mamu-235

A*01+ rhesus monkeys were inoculated intravenously at doses ranging from 10

6 to 10

9, 236

using two animals per dose. Twenty-three weeks after rBCG priming, the two monkeys 237

that were immunized intradermally received a second injection intradermally with 107 cfu 238

rBCG, and those that were immunized intravenously were injected again with the same 239

vaccine doses. No adverse effects were observed in any of these monkeys after the 240

second rBCG administration. However, transient leukocytosis and lymphocytosis were 241

observed in monkeys that received two inoculations of 109 cfu rBCG intravenously. All 242

ten rBCG-immunized animals and three naive control animals were then vaccinated with 243

1010

particles rAd5 expressing SIV Gag/Pol and 1010

particles rAd5 expressing SIV Env 244

43 weeks after the first rBCG vaccination. 245

246

BCG vector-specific immune responses after heterologous priming with 247

recombinant BCG and boosting with rAdenovirus expressing SIV Gag/Pol and Env 248

proteins. We first assessed BCG vector-specific immune responses in the immunized and 249

control animals. As shown in Fig. 3, BCG PPD cellular responses were seen as early as 250

one week after rBCG inoculation. By week 2, strong PPD responses (greater than 3000 251

IFNγ-producing cells) were observed, and this level of immune response was sustained in 252

the ensuing weeks following immunization. Interestingly, the second immunization with 253

rBCG resulted in a robust recall response to the BCG vector. Peak responses were seen 254


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

one week after homologous boosting, with a mean greater than the maximum level of 255

detection of 5000 IFNγ-producing cells. In the ensuing weeks, this cellular immune 256

response contracted gradually. As expected, there were no increases in the frequency of 257

peripheral blood IFNγ-producing cells in response to PPD stimulation following rAd5 258

immunization. Moreover, monkeys that were immunized with rAd5 alone did not 259

develop PPD-specific cellular responses. Taken together, these results suggest that rBCG 260

immunization elicits a strong cellular response to the BCG vector that can be boosted by 261

repeated BCG administration. 262

263

SIV-specific T cell responses following heterologous rBCG prime, rAd5 boost 264

vaccination. Following a single or repeated immunization with recombinant BCG 265

expressing SIV Gag, Pol and Env, rhesus monkeys developed low but detectible SIV-266

specific T cell responses. Peripheral IFNγ ELISpot responses to Gag and Pol were for the 267

most part below 100 spot-forming cells, and responses to Env were negligible. 268

Furthermore, SIV Gag p11C tetramer-binding CD8+ T cells were detected only after in 269

vitro stimulation of monkey peripheral blood lymphocytes with the Gag p11C peptide 270

and IL-2 (Fig. 4). Heterologous immunization of the rBCG-primed monkeys with rAd5 271

constructs expressing SIV Gag/Pol and Env generated a high frequency of IFNγ-272

producing T cells in response to stimulation with the immunodominant Gag p11C peptide 273

as well as Gag and Pol peptide pools, but not to the Env peptide pool (Fig. 5). The Gag- 274

and Pol-specific cellular responses were higher than those in rAd5-immunized control 275

animals. T cell responses in rBCG/rAd5-vaccinated animals peaked 2 to 4 weeks after 276

rAd5 vaccination and subsequently contracted over time. SIV-specific cellular responses 277

remained higher in rBCG primed animals than in the control animals (Fig. 5). 278


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

rBCG-primed monkeys also developed robust expansions of p11C-specific CD8+ 279

T cells compared to the control monkeys following rAd5 administration (Fig. 6). The 280

frequency of tetramer-binding CD8+ T cells peaked 2 to 4 weeks after rAd5 281

administration and declined in the ensuing weeks. At the peak immune response, there 282

was a trend toward an association between the dose of the rBCG priming immunogen and 283

the magnitude of the vaccine-elicited T cell response (Fig. 7). This association did not 284

achieve statistical significance in the Kruskal-Wallis test adjusted for multiple 285

comparisons because of the small number of monkeys studied at each dose. The mean 286

frequency of IFNγ-producing cells increased as the dose of the rBCG priming 287

immunogen increased. Statistical analysis using the Mann-Whitney test showed that Gag 288

ELISpot and Gag p11C-tetramer responses were higher after rAd5 administration in the 289

rBCG primed than in the control animals (Fig. 8A and B). While at week 4 after rAd5 290

immunization a trend toward higher mean cellular immune responses in rBCG/rAd 291

vaccinated animals than in control animals was observed, the difference between the 292

responses in the rBCG/rAd5 and the rAd5 alone vaccinated monkeys achieved statistical 293

significance at weeks 2, 10, and 21 (Fig. 8A and B). The results of these immunogenicity 294

studies suggest that recombinant BCG vaccines can prime effectively for a recombinant 295

Ad5 vector boost in rhesus monkeys. 296

297

Functional profiles of SIV-specific T cell responses after rBCG/rAd vaccination in 298

rhesus monkeys. Studies suggest that both the magnitude and the quality of the cellular 299

immune response contribute to the control of immunodeficiency virus infections (3, 40). 300

We therefore assessed the functional profile of the cellular immune responses elicited by 301

the rBCG prime/rAd5 boost vaccination regimen. Peripheral blood lymphocytes isolated 302


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

from rBCG primed/rAd5 or rAd5 only vaccinated monkeys were stimulated with 303

overlapping 15-mer peptides spanning the SIV Gag protein, and production of IFNγ, 304

TNFα, and IL-2 by T cells was measured by intracellular cytokine staining. The total 305

production of these three cytokines by either CD4+ or CD8

+ T cells is shown (Fig. 9A, 306

upper panel). Monkeys receiving the rBCG/rAd5 vector vaccine regimen generated 307

higher T cell responses than those vaccinated with rAd5 alone. Moreover, the cytokine 308

responses were biased toward CD8+ T cells regardless of vaccination strategy. 309

We next assessed SIV Gag pooled peptide-stimulated production of these three 310

cytokines to characterize the functional profiles of the vaccine-elicited T cell responses. 311

Total SIV-specific cytokine-producing CD8+ T cells were divided into seven distinct 312

populations based on their production of IFNγ, TNFα, and IL-2, either individually or in 313

combination. The profiles of the functional capacities of the cells are shown by 314

expressing each type of cytokine response as a proportion of the total response. The 315

mean values for the animals in each vaccine group are shown in a series of pie charts 316

(Fig. 9A, lower panel). 317

The SIV-specific CD8+ T cell responses induced by either rBCG/rAd5 or rAd5 318

alone vaccination were highly polyfunctional. The predominant population of 319

polyfunctional T cells generated in the vaccinated monkeys produced both IFNγ and 320

TNFα combined. A small fraction of the polyfunctional T cells were also capable of 321

producing IL-2 in conjunction with the other cytokines. Interestingly, the overall 322

functional profile of the CD8+ T cell response was similar in both rBCG/rAd5 and rAd5 323

only vaccinated animals. 324

Finally, the CD8+ T cell response specific for the immunodominant p11C Gag 325

epitope peptide was assessed. Higher percentages of p11C tetramer-binding CD8+ T cells 326


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

were seen in monkeys immunized with rBCG/rAd5 than in those immunized with rAd5 327

alone (Fig. 9B). However, the proportion of tetramer-binding CD8+ T cells expressing the 328

memory-associated molecule CD28 as well as producing IFNγ, TNFα, and IL-2 was 329

similar in both the rBCG/rAd5 and the rAd5 alone groups. Taken together, these findings 330

show that rBCG prime/rAd5 boost vaccination generated large magnitude T cell 331

responses with a polyfunctional phenotype. In addition, rBCG priming did not influence 332

the functional profile of the rAd5 vaccine-elicited T cells. 333

334

335


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

Discussion 336

337

A prime/boost vaccination strategy using recombinant BCG as the priming vector 338

and recombinant viruses or protein as the boosting immunogen has been demonstrated to 339

be very effective at eliciting antigen-specific immune responses (4, 27, 31). In the present 340

vaccine immunogenicity study, we found that priming with recombinant BCG and 341

subsequently boosting with recombinant adenovirus expressing SIV antigens linked to 342

the M. tuberculosis 19kDa signal peptide generated robust and polyfunctional SIV-343

specific T cell responses in vaccinated rhesus monkeys. 344

Following rBCG vaccination, mycobacteria PPD-stimulated IFNγ ELISpot 345

responses were strong; however, cellular responses specific for the SIV antigens were 346

modest at best. A second inoculation with rBCG did not enhance SIV-specific T cell 347

responses in the animals. These results are consistent with previous studies suggesting 348

that transgenes expressed in recombinant BCG are marginally immunogenic (2, 9). While 349

an explanation for the low ELISpot responses elicited by rBCG vaccines is not entirely 350

clear, it may be a result of low transgene expression or inefficient presentation of the 351

transgene product to T cells (9). 352

When rBCG-primed rhesus monkeys were boosted with rAd5 expressing the 353

same SIV antigens, we saw a substantial enhancement of durable SIV Gag and Pol-354

specific cellular responses as determined by SIV Gag-p11C tetramer and by IFNγ 355

ELISpot responses. We did not evaluate the epitopic breadth of these vaccine elicited T 356

cells. Cellular immune responses to SIV Env, however, were comparable in the rBCG 357

primed and naïve monkeys. The absence of priming for cellular responses to this viral 358

antigen is likely due to the instability of the envelope protein expressed in mycobacteria. 359

We found that the mycobacteria pJH222 plasmid with the SIV env gene insert was 360


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

inefficient at transforming BCG and the few transformants that grew either did not 361

express SIV gp120 protein or expressed SIV gp120 protein at very low levels. 362

Importantly, however, the absence of envelope priming in monkeys that received this 363

vaccine construct argues against the possibility that the recombinant BCG immunogen 364

primed for enhanced secondary immune responses by nonspecific activation of the 365

immune system. 366

Although recombinant mycobacteria-induced CD8+ T cell responses are of lower 367

magnitude than the responses induced by DNA and other vaccine vectors used as priming 368

immunogens, recombinant mycobacteria-primed T cells have a tremendous capacity for 369

secondary expansion in the setting of heterologous prime/boost vaccination (15). We and 370

others have shown that recombinant BCG and other mycobacteria can induce central 371

memory CD8+ T cell differentiation in vivo (6, 15, 42). The ability of BCG and other 372

mycobacteria to generate memory CD8+ T cells could be due to the propensity of 373

mycobacteria to stimulate CD4+ T helper cells, which has been shown to be important for 374

driving memory CD8+ T cell differentiation (37, 39). 375

It has been suggested that the functional heterogeneity of T cell responses may be 376

associated with successful containment of microbial infections. The extent of T cell 377

polyfunctionality has been correlated with protection against leishmaniasis in mice, HIV-378

1 in humans (1, 3, 8, 10, 32, 41), and SIV in nonhuman primates (13). We previously 379

showed that the magnitude and polyfunctionality of virus-specific CD8+ T cell responses 380

were associated with control of viral replication following SHIV-89.6P challenge in 381

rhesus monkeys (40). In the present study using intracellular cytokine staining and 382

polychromatic flow cytometry, we found a higher frequency of cytokine-producing cells 383

in rBCG primed monkeys than in control monkeys following rAd5 boost. There was also 384


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

a CD8+ T cell bias in these responses. However, both rBCG/rAd5 and rAd5 alone 385

vaccination generated T cell responses with similar functional profiles. In both vaccinated 386

groups of monkeys, over half of the virus-specific CD8+ T cells were polyfunctional, 387

capable of producing two or more of the cytokines IL-2, IFNγ, and TNFα. In addition, the 388

proportion of p11C-specific CD8+ T cells expressing CD28 and the three cytokines was 389

similar in both groups of animals. Phenotypically, the majority of the cytokine-producing 390

CD8+ T cells were capable of secreting both TNFα and IFNγ and significant populations 391

of these cells were able to secrete all three or the combination of IFNγ and IL-2 after Gag 392

peptide stimulation. These IL-2-producing cells are likely long-lived central memory T 393

cells that are capable of rapid expansion after re-exposure to cognate antigen (19, 35). 394

The finding of similar functional profiles of vaccine-elicited T cells in the rAd5 alone and 395

rBCG prime/rAd5 boost immunized monkeys was certainly unexpected in view of the 396

reports of a change in the profiles of these cells mediated by a plasmid DNA priming 397

immunization. 398

Results from the recent STEP clinical vaccine trial showed that rAd5 as a stand-399

alone vaccine was not effective against HIV-1 (5, 26). Moreover, this trial suggested that 400

rAd5 may enhance HIV-1 acquisition in individuals with pre-existing Ad5 immunity. 401

However, rare serotype rAd vectors or recombinant poxvirus vectors combined with 402

other rAd serotypes or other vectors such DNA or BCG in a prime/boost vaccine regimen 403

could potentially confer some protection against an AIDS virus infection since these 404

immunization regimens generate robust anti-HIV-1 immune responses (22). The present 405

study provides further evidence for the potential utility of rBCG immunogens for 406

inducing HIV-1-specific immune responses. 407

408


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

Acknowledgements 410

411

Funding for this work was made possible through the CAVD program of the Bill and 412

Melinda Gates Foundation. 413

414

415


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

Figure Legends 416

417

FIG. 1. Immunization schedule. Ten MamuA*01+ rhesus monkeys were immunized at 418

weeks 0 and 23 with recombinant BCG expressing SIV Gag, Pol and Env at the indicated 419

doses (106, 10

7, 10

8, or 10

9 cfu) either intradermally or intravenously. All monkeys were 420

subsequently boosted intramuscularly (IM) with 1010

vp rAd5 expressing SIV Gag/Pol 421

and Env at week 43. Three control MamuA*1+ monkeys were included that were 422

immunized intramuscularly with 1010

vp rAd5 expressing Gag/Pol and Env alone. 423

424

FIG. 2. Recombinant BCG constructs expressing SIV Gag, Pol or Env. BCG Pasteur 425

was transformed with the multi-copy pJH222 plasmid containing either the SIV gag, pol 426

or env gene. Expression of the SIV antigens were analyzed by Western blotting of whole 427

bacterial cell lysates using an anti-HA mAb. Each lane represents an individual rBCG 428

transformant and arrows mark the putative expression of the full-length SIV antigens. 429

Selected clones expressing either the SIV Gag, Pol or Env proteins were selected for 430

immunization into rhesus monkeys (denoted with an asterisk). 431

432

FIG. 3. BCG PPD-specific IFNγ ELISpot responses in PBMC from rhesus monkeys 433

following rBCG immunization. BCG vector-specific responses were assessed in 13 434

rhesus monkeys following immunization. Freshly isolated monkey PBMC were co-435

cultured with 25 µg/ml bovine PPD overnight, and cellular production of IFNγ was 436

ascertained using an IFNγ ELISpot assay. Results presented are the mean (± SEM) 437

number of IFNγ spot-forming cells (SFC) per 106 PBMC from the 10 rhesus monkeys 438

immunized at week 0 and 23 with rBCG either intradermally or intravenously, followed 439


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

by intramuscular injection with rAd SIV Gag/Pol and Env (rBCG group represented as 440

filled-in circles), and from the 3 monkeys that were immunized only with the rAd5 441

Gag/Pol and Env vectors (Control group represented as open squares). 442

443

FIG. 4. Vaccine-elicited tetramer-binding CD8+ T cell responses after in vitro culture of 444

PBMC from rhesus monkeys following rBCG priming. SIV Gag p11C-tetramer 445

responses were assessed after priming with rBCG expressing SIV Gag, Pol, and Env 446

immunogens. PBMC was isolated from the ten rBCG-inoculated rhesus monkeys before 447

(prebleed), four weeks after the first immunization (wk 4) and four weeks after the 448

second immunization (wk 27) with recombinant BCG. PBMC were cultured with 1 µg/ml 449

of p11C peptide for 10 to 13 days and supplementation with 20U/ml of IL-2 starting from 450

day 3 of the culture. T cells were then stained with the p11C tetramer and analyzed by 451

flow cytometry. Results shown are % p11C tetramer+ CD8

+ T cells for each vaccinated 452

animal. 453

454

FIG. 5. The magnitude and kinetics of the SIV-specific IFNγ ELISpot responses in 455

PBMC from rhesus monkeys after immunization with rBCG SIV Gag, Pol and Env and 456

rAd SIV Gag/Pol and rAd SIV Env vectors. Freshly isolated PBMC from immunized 457

monkeys were cultured with SIV Gag, Pol and Env peptide pools or the Gag p11C 458

peptide, and IFNγ producing-cells were measured using an IFNγ ELISpot assay. Data 459

shown are the mean (± SEM) IFNγ SFC per 106 PBMC for the 10 rBCG/rAd5 (filled-in 460

circles) and 3 rAd5 alone rBCG/rAd5 (open squares) immunized macaques. Arrows 461

indicate the timepoints of immunization: rBCG immunization at weeks 0 and 23; rAd5 462

immunization at week 43. 463


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

464

FIG. 6. Vaccine-elicited tetramer-binding CD8+ T cell responses in peripheral blood 465

from rhesus monkeys following rAd5 boosting. Ten rBCG/rAd5 (filled-in circles) and 3 466

rAd5 alone (open squares) immunized monkeys were studied. Peripheral blood SIV Gag 467

p11C-specific T cells were assessed by staining with the p11C tetramer. Data shown are 468

the mean (± SEM) % tetramer-binding CD8+ T cells at the indicated times following 469

rBCG and rAd5 immunizations. 470

471

FIG. 7. Dose-dependent trend of peak SIV Gag-specific IFNγ ELISpot responses 472

following rAd5 boosting of rBCG-primed rhesus monkeys in comparison to rAd5 only 473

immunized macaques. PBMC were isolated from rhesus monkeys at the time of the peak 474

of immune responses, four weeks after boosting with rAd5 Gag/Pol and rAd5 Env 475

vectors, respectively two weeks after immunizing with rAd5 alone (control). The doses of 476

the preceding rBCG prime (ID or IV) are indicated. PBMC were stimulated with SIV 477

Gag pooled peptides, and IFNγ ELISpot assays were performed to measure SIV Gag-478

specific cellular immune responses. The data shown are the IFNγ SFC per 106 PBMC for 479

each individual monkey as well as the median Gag-specific ELISpot responses in the 480

PBMC of the 10 monkeys primed with the indicated dose of rBCG and boosted with the 481

rAd5 SIV Gag/Pol and Env vectors (filled-in shapes), and of the 3 monkeys receiving 482

only the rAd5 SIV Gag/Pol and Env vectors (open squares – control). 483

484

FIG. 8. Comparison of SIV Gag-specific T cell responses following rAd5 SIV Gag/Pol 485

boosting of monkeys primed with rBCG and SIV Gag-specific T cell responses following 486

only rAd5 immunization. Freshly isolated PBMC were assayed for Gag-specific IFNγ 487


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

production using an ELISpot assay (A) and SIV Gag-specific T cells by staining with a 488

p11C tetramer (B). Results shown are the distribution of the numbers of IFNγ SFC per 489

106 PBMC (A) or % tetramer-binding CD8

+ T cells (B) for each vaccinated monkey at 490

the indicated times following recombinant Ad5 vector boost. Error bars indicate the 491

median and interquartile range. The two-sided nonparametric Mann-Whitney test was 492

used to determine the P value of the difference in T cell responses between the rBCG 493

primed/rAd5 boosted animals (filled-in circles - rBCG) and the rAd5 vector only 494

immunized animals (open squares - control). 495

496

FIG. 9. Heterologous rBCG prime/rAd5 boost immunization elicits polyfunctional SIV 497

Gag-specific CD8+ T cells. PBMC were isolated from rhesus monkeys 13 weeks after 498

boosting rBCG-primed animals with rAd5 SIV Gag/Pol. The PBMC were exposed to 499

pools of overlapping peptides spanning the Gag protein, and cytokine production was 500

measured by mAb staining and flow cytometric analysis. (A) The median % and 501

interquartile range of the total cytokine production by CD4+ and CD8

+ T cells is shown. 502

The antigen-specific CD8+ T cells were divided into seven distinct populations based on 503

their production of IFNγ, TNFα, and IL-2. The cytokine profiles were determined by 504

expressing each cytokine response as a proportion of the total antigen-specific cytokine-505

producing CD8+ T cell response. Data were analyzed using the SPICE software and are 506

presented as the mean values from the 10 animals of the rBCG-primed/rAd5-boosted 507

group (rBCG) and 3 animals of the rAd5 only immunized group (Control) in a pie chart. 508

(B) The cytokine production capacity and CD28 expression of the SIV Gag p11C-509

specific CD8+ T cells were assessed. PBMC isolated from rhesus monkeys 13 weeks after 510

rAd5 SIV Gag/Pol immunization were stained with the p11C tetramer and anti-CD28 511


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

mAb. These PBMC were also stimulated with the p11C peptide, and cytokine production 512

was measured by staining with mAb and analyzed by flow cytometry. Data shown are the 513

median (with interquartile range) % of the total CD8+ T cells that bound the p11C 514

tetramer and % p11C tetramer+ CD8

+ T cells (p11C

+CD8

+ T cells) that expressed CD28 515

or produced IFNγ, TNFα or IL-2. 516

517

518


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

References 519

520

1. Almeida, J. R., D. A. Price, L. Papagno, Z. A. Arkoub, D. Sauce, E. 521

Bornstein, T. E. Asher, A. Samri, A. Schnuriger, I. Theodorou, D. 522

Costagliola, C. Rouzioux, H. Agut, A. G. Marcelin, D. Douek, B. Autran, and 523 V. Appay. 2007. Superior control of HIV-1 replication by CD8+ T cells is 524

reflected by their avidity, polyfunctionality, and clonal turnover. J Exp Med 525

204:2473-85. 526

2. Ami, Y., Y. Izumi, K. Matsuo, K. Someya, M. Kanekiyo, S. Horibata, N. 527

Yoshino, K. Sakai, K. Shinohara, S. Matsumoto, T. Yamada, S. Yamazaki, N. 528 Yamamoto, and M. Honda. 2005. Priming-boosting vaccination with 529

recombinant Mycobacterium bovis bacillus Calmette-Guerin and a nonreplicating 530

vaccinia virus recombinant leads to long-lasting and effective immunity. J Virol 531

79:12871-9. 532

3. Betts, M. R., M. C. Nason, S. M. West, S. C. De Rosa, S. A. Migueles, J. 533

Abraham, M. M. Lederman, J. M. Benito, P. A. Goepfert, M. Connors, M. 534 Roederer, and R. A. Koup. 2006. HIV nonprogressors preferentially maintain 535

highly functional HIV-specific CD8+ T cells. Blood 107:4781-9. 536

4. Brandt, L., Y. A. W. Skeiky, M. R. Alderson, Y. Lobet, W. Dalemans, O. C. 537

Turner, R. J. Basaraba, A. A. Izzo, T. M. Lasco, P. L. Chapman, S. G. Reed, 538 and I. M. Orme. 2004. The Protective Effect of the Mycobacterium bovis BCG 539

Vaccine Is Increased by Coadministration with the Mycobacterium tuberculosis 540

72-Kilodalton Fusion Polyprotein Mtb72F in M. tuberculosis-Infected Guinea 541

Pigs. Infect. Immun. 72:6622-6632. 542

5. Buchbinder, S. P., D. V. Mehrotra, A. Duerr, D. W. Fitzgerald, R. Mogg, D. 543

Li, P. B. Gilbert, J. R. Lama, M. Marmor, C. Del Rio, M. J. McElrath, D. R. 544

Casimiro, K. M. Gottesdiener, J. A. Chodakewitz, L. Corey, and M. N. 545 Robertson. 2008. Efficacy assessment of a cell-mediated immunity HIV-1 546

vaccine (the Step Study): a double-blind, randomised, placebo-controlled, test-of-547

concept trial. Lancet 372:1881-93. 548

6. Cayabyab, M. J., A. H. Hovav, T. Hsu, G. R. Krivulka, M. A. Lifton, D. A. 549

Gorgone, G. J. Fennelly, B. F. Haynes, W. R. Jacobs, Jr., and N. L. Letvin. 550 2006. Generation of CD8+ T-cell responses by a recombinant nonpathogenic 551

Mycobacterium smegmatis vaccine vector expressing human immunodeficiency 552

virus type 1 Env. J Virol 80:1645-52. 553

7. Connell, N. D., E. Medina-Acosta, W. R. McMaster, B. R. Bloom, and D. G. 554

Russell. 1993. Effective immunization against cutaneous leishmaniasis with 555

recombinant bacille Calmette-Guerin expressing the Leishmania surface 556

proteinase gp63. Proc Natl Acad Sci U S A 90:11473-7. 557

8. Darrah, P. A., D. T. Patel, P. M. De Luca, R. W. Lindsay, D. F. Davey, B. J. 558

Flynn, S. T. Hoff, P. Andersen, S. G. Reed, S. L. Morris, M. Roederer, and R. 559 A. Seder. 2007. Multifunctional TH1 cells define a correlate of vaccine-mediated 560

protection against Leishmania major. Nat Med 13:843-50. 561


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

9. Dennehy, M., and A.-L. Williamson. 2005. Factors influencing the immune 562

response to foreign antigen expressed in recombinant BCG vaccines. Vaccine 563

23:1209-1224. 564

10. Douek, D. C., M. Roederer, and R. A. Koup. 2008. Emerging Concepts in the 565

Immunopathogenesis of AIDS. Annu Rev Med. 566

11. Edelman, R., K. Palmer, K. G. Russ, H. P. Secrest, J. A. Becker, S. A. 567

Bodison, J. G. Perry, A. R. Sills, A. G. Barbour, C. J. Luke, M. S. Hanson, C. 568 K. Stover, J. E. Burlein, G. P. Bansal, E. M. Connor, and S. Koenig. 1999. 569

Safety and immunogenicity of recombinant Bacille Calmette-Guerin (rBCG) 570

expressing Borrelia burgdorferi outer surface protein A (OspA) lipoprotein in 571

adult volunteers: a candidate Lyme disease vaccine. Vaccine 17:904-14. 572

12. Fennelly, G. J., J. L. Flynn, V. ter Meulen, U. G. Liebert, and B. R. Bloom. 573

1995. Recombinant bacille Calmette-Guerin priming against measles. J Infect Dis 574

172:698-705. 575

13. Genesca, M., T. Rourke, J. Li, K. Bost, B. Chohan, M. B. McChesney, and C. 576

J. Miller. 2007. Live attenuated lentivirus infection elicits polyfunctional simian 577

immunodeficiency virus Gag-specific CD8+ T cells with reduced apoptotic 578

susceptibility in rhesus macaques that control virus replication after challenge 579

with pathogenic SIVmac239. J Immunol 179:4732-40. 580

14. Goonetilleke, N. P., H. McShane, C. M. Hannan, R. J. Anderson, R. H. 581

Brookes, and A. V. Hill. 2003. Enhanced immunogenicity and protective 582

efficacy against Mycobacterium tuberculosis of bacille Calmette-Guerin vaccine 583

using mucosal administration and boosting with a recombinant modified vaccinia 584

virus Ankara. J Immunol 171:1602-9. 585

15. Hovav, A.-H., M. J. Cayabyab, M. W. Panas, S. Santra, J. Greenland, R. 586

Geiben, B. F. Haynes, W. R. Jacobs, Jr., and N. L. Letvin. 2007. Rapid 587

Memory CD8+ T-Lymphocyte Induction through Priming with Recombinant 588

Mycobacterium smegmatis. J. Virol. 81:74-83. 589

16. Kremer, L., G. Riveau, A. Baulard, A. Capron, and C. Locht. 1996. 590

Neutralizing antibody responses elicited in mice immunized with recombinant 591

bacillus Calmette-Guerin producing the Schistosoma mansoni glutathione S-592

transferase. J Immunol 156:4309-17. 593

17. Kuroda, M. J., J. E. Schmitz, D. H. Barouch, A. Craiu, T. M. Allen, A. Sette, 594

D. I. Watkins, M. A. Forman, and N. L. Letvin. 1998. Analysis of Gag-specific 595

cytotoxic T lymphocytes in simian immunodeficiency virus-infected rhesus 596

monkeys by cell staining with a tetrameric major histocompatibility complex class 597

I-peptide complex. J Exp Med 187:1373-81. 598

18. Langermann, S., S. R. Palaszynski, J. E. Burlein, S. Koenig, M. S. Hanson, D. 599

E. Briles, and C. K. Stover. 1994. Protective humoral response against 600

pneumococcal infection in mice elicited by recombinant bacille Calmette-Guerin 601

vaccines expressing pneumococcal surface protein A. J Exp Med 180:2277-86. 602

19. Leone, A., L. J. Picker, and D. L. Sodora. 2009. IL-2, IL-7 and IL-15 as 603

immuno-modulators during SIV/HIV vaccination and treatment. Curr HIV Res 604

7:83-90. 605

20. Letvin, N. L., Y. Huang, B. K. Chakrabarti, L. Xu, M. S. Seaman, K. 606

Beaudry, B. Korioth-Schmitz, F. Yu, D. Rohne, K. L. Martin, A. Miura, W. 607

P. Kong, Z. Y. Yang, R. S. Gelman, O. G. Golubeva, D. C. Montefiori, J. R. 608


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

Mascola, and G. J. Nabel. 2004. Heterologous envelope immunogens contribute 609

to AIDS vaccine protection in rhesus monkeys. J Virol 78:7490-7. 610

21. Letvin, N. L., J. R. Mascola, Y. Sun, D. A. Gorgone, A. P. Buzby, L. Xu, Z. Y. 611

Yang, B. Chakrabarti, S. S. Rao, J. E. Schmitz, D. C. Montefiori, B. R. 612 Barker, F. L. Bookstein, and G. J. Nabel. 2006. Preserved CD4+ central 613

memory T cells and survival in vaccinated SIV-challenged monkeys. Science 614

312:1530-3. 615

22. Liu, J., K. L. O/'Brien, D. M. Lynch, N. L. Simmons, A. La Porte, A. M. 616

Riggs, P. Abbink, R. T. Coffey, L. E. Grandpre, M. S. Seaman, G. Landucci, 617

D. N. Forthal, D. C. Montefiori, A. Carville, K. G. Mansfield, M. J. Havenga, 618 M. G. Pau, J. Goudsmit, and D. H. Barouch. 2008. Immune control of an SIV 619

challenge by a T-cell-based vaccine in rhesus monkeys. Nature. 620

23. Mascola, J. R., A. Sambor, K. Beaudry, S. Santra, B. Welcher, M. K. Louder, 621

T. C. Vancott, Y. Huang, B. K. Chakrabarti, W. P. Kong, Z. Y. Yang, L. Xu, 622 D. C. Montefiori, G. J. Nabel, and N. L. Letvin. 2005. Neutralizing antibodies 623

elicited by immunization of monkeys with DNA plasmids and recombinant 624

adenoviral vectors expressing human immunodeficiency virus type 1 proteins. J 625

Virol 79:771-9. 626

24. Matsumoto, S., H. Yukitake, H. Kanbara, and T. Yamada. 1998. 627

Recombinant Mycobacterium bovis bacillus Calmette-Guerin secreting merozoite 628

surface protein 1 (MSP1) induces protection against rodent malaria parasite 629

infection depending on MSP1-stimulated interferon gamma and parasite-specific 630

antibodies. J Exp Med 188:845-54. 631

25. Mattapallil, J. J., D. C. Douek, A. Buckler-White, D. Montefiori, N. L. 632

Letvin, G. J. Nabel, and M. Roederer. 2006. Vaccination preserves CD4 633

memory T cells during acute simian immunodeficiency virus challenge. J Exp 634

Med 203:1533-41. 635

26. McElrath, M. J., S. C. De Rosa, Z. Moodie, S. Dubey, L. Kierstead, H. Janes, 636

O. D. Defawe, D. K. Carter, J. Hural, R. Akondy, S. P. Buchbinder, M. N. 637

Robertson, D. V. Mehrotra, S. G. Self, L. Corey, J. W. Shiver, and D. R. 638 Casimiro. 2008. HIV-1 vaccine-induced immunity in the test-of-concept Step 639

Study: a case-cohort analysis. Lancet 372:1894-905. 640

27. McShane, H., and A. Hill. 2005. Prime-boost immunisation strategies for 641

tuberculosis. Microbes Infect 7:962-7. 642

28. McShane, H., A. A. Pathan, C. R. Sander, S. M. Keating, S. C. Gilbert, K. 643

Huygen, H. A. Fletcher, and A. V. Hill. 2004. Recombinant modified vaccinia 644

virus Ankara expressing antigen 85A boosts BCG-primed and naturally acquired 645

antimycobacterial immunity in humans. Nat Med 10:1240-4. 646

29. Nascimento, I. P., W. O. Dias, R. P. Mazzantini, E. N. Miyaji, M. Gamberini, 647

W. Quintilio, V. C. Gebara, D. F. Cardoso, P. L. Ho, I. Raw, N. Winter, B. 648 Gicquel, R. Rappuoli, and L. C. Leite. 2000. Recombinant Mycobacterium 649

bovis BCG expressing pertussis toxin subunit S1 induces protection against an 650

intracerebral challenge with live Bordetella pertussis in mice. Infect Immun 651

68:4877-83. 652

30. National. 1996. Guide for the care and use of laboratory animals National 653

Academic Press, Washington, D.C. 654


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

31. Orme, I. M. 2005. Current progress in tuberculosis vaccine development. 655

Vaccine. Vaccines and Immunisation. Based on the Fourth World Congress on 656

Vaccines and Immunisation 23:2105-2108. 657

32. Peretz, Y., M. L. Ndongala, S. Boulet, M. R. Boulassel, D. Rouleau, P. Cote, 658

D. Longpre, J. P. Routy, J. Falutz, C. Tremblay, C. M. Tsoukas, R. P. Sekaly, 659 and N. F. Bernard. 2007. Functional T cell subsets contribute differentially to 660

HIV peptide-specific responses within infected individuals: correlation of these 661

functional T cell subsets with markers of disease progression. Clin Immunol 662

124:57-68. 663

33. Romano, M., S. D'Souza, P. Y. Adnet, R. Laali, F. Jurion, K. Palfliet, and K. 664

Huygen. 2006. Priming but not boosting with plasmid DNA encoding mycolyl-665

transferase Ag85A from Mycobacterium tuberculosis increases the survival time 666

of Mycobacterium bovis BCG vaccinated mice against low dose intravenous 667

challenge with M. tuberculosis H37Rv. Vaccine 24:3353-64. 668

34. Russell, M. S., M. Iskandar, O. L. Mykytczuk, J. H. E. Nash, L. Krishnan, 669

and S. Sad. 2007. A Reduced Antigen Load In Vivo, Rather Than Weak 670

Inflammation, Causes a Substantial Delay in CD8+ T Cell Priming against 671

Mycobacterium bovis (Bacillus Calmette-Guerin). J Immunol 179:211-220. 672

35. Sallusto, F., J. Geginat, and A. Lanzavecchia. 2004. Central memory and 673

effector memory T cell subsets: function, generation, and maintenance. Annu Rev 674

Immunol 22:745-63. 675

36. Santosuosso, M., S. McCormick, X. Zhang, A. Zganiacz, and Z. Xing. 2006. 676

Intranasal boosting with an adenovirus-vectored vaccine markedly enhances 677

protection by parenteral Mycobacterium bovis BCG immunization against 678

pulmonary tuberculosis. Infect Immun 74:4634-43. 679

37. Shedlock, D. J., and H. Shen. 2003. Requirement for CD4 T cell help in 680

generating functional CD8 T cell memory. Science 300:337-9. 681

38. Stover, C. K., G. P. Bansal, M. S. Hanson, J. E. Burlein, S. R. Palaszynski, J. 682

F. Young, S. Koenig, D. B. Young, A. Sadziene, and A. G. Barbour. 1993. 683

Protective immunity elicited by recombinant bacille Calmette-Guerin (BCG) 684

expressing outer surface protein A (OspA) lipoprotein: a candidate Lyme disease 685

vaccine. J Exp Med 178:197-209. 686

39. Sun, J. C., and M. J. Bevan. 2003. Defective CD8 T cell memory following 687

acute infection without CD4 T cell help. Science 300:339-42. 688

40. Sun, Y., S. Santra, J. E. Schmitz, M. Roederer, and N. L. Letvin. 2008. 689

Magnitude and quality of vaccine-elicited T-cell responses in the control of 690

immunodeficiency virus replication in rhesus monkeys. J Virol 82:8812-9. 691

41. Turk, G., M. M. Gherardi, N. Laufer, M. Saracco, R. Luzzi, J. H. Cox, P. 692

Cahn, and H. Salomon. 2008. Magnitude, breadth, and functional profile of T-693

cell responses during human immunodeficiency virus primary infection with B 694

and BF viral variants. J Virol 82:2853-66. 695

42. van Faassen, H., M. Saldanha, D. Gilbertson, R. Dudani, L. Krishnan, and S. 696

Sad. 2005. Reducing the Stimulation of CD8+ T Cells during Infection with 697

Intracellular Bacteria Promotes Differentiation Primarily into a Central 698

(CD62LhighCD44high) Subset. J Immunol 174:5341-5350. 699

43. Vordermeier, H. M., S. G. Rhodes, G. Dean, N. Goonetilleke, K. Huygen, A. 700

V. Hill, R. G. Hewinson, and S. C. Gilbert. 2004. Cellular immune responses 701


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

induced in cattle by heterologous prime-boost vaccination using recombinant 702

viruses and bacille Calmette-Guerin. Immunology 112:461-70. 703

44. Vuola, J. M., S. Keating, D. P. Webster, T. Berthoud, S. Dunachie, S. C. 704

Gilbert, and A. V. S. Hill. 2005. Differential Immunogenicity of Various 705

Heterologous Prime-Boost Vaccine Regimens Using DNA and Viral Vectors in 706

Healthy Volunteers. J Immunol 174:449-455. 707

45. Williams, A., N. P. Goonetilleke, H. McShane, S. O. Clark, G. Hatch, S. C. 708

Gilbert, and A. V. Hill. 2005. Boosting with poxviruses enhances 709

Mycobacterium bovis BCG efficacy against tuberculosis in guinea pigs. Infect 710

Immun 73:3814-6. 711

712

713


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

Recombinant BCG prime-recombinant adenovirus boost...

Documents

Transcript of Recombinant BCG prime-recombinant adenovirus boost...