Strong HIV-Specific CD4+ and CD8+ T lymphocyte proliferative responses in healthy 1

individuals immunized with a HIV-1 DNA vaccine and boosted with HIV-1 recombinant 2

Modified Vaccinia virus Ankara (MVA) 3

4

Said Aboud1,2‡, Charlotta Nilsson2,3‡, Katarina Karlén3, Mary Marovich4, Britta Wahren2,3, 5

Eric Sandström5, Hans Gaines2,3, Gunnel Biberfeld2,3, Karina Godoy-Ramirez3* 6

7

‡Authors contributed equally to this report 8

1Muhimbili University of Health and Allied Sciences, Department of Microbiology and 9

Immunology, Dar es Salaam, Tanzania 10

2Department of Microbiology, Tumor and Cell biology, Karolinska Institutet, Stockholm, 11

Sweden 12

3Swedish Institute for Infectious Disease Control, Solna, Sweden 13

4Walter Reed Army Institute for Research, Department of Retrovirology, Rockville, 14

Maryland, USA 15

5Venhälsan, Department of Education and Clinical Research, Karolinska Institutet, 16

Södersjukhuset, Stockholm, Sweden. 17

18

Running title: HIV-1 vaccine-induced T-lymphocyte proliferation 19

20

Corresponding author: 21

*Karina Godoy-Ramirez, Swedish Institute for Infectious Disease control, SE-171 82 Solna, 22

Sweden. Phone: +46 8 457 2668. Fax: +46 8 33 74 60. E-mail: karina.godoy@smi.se 23

24

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.00008-10 CVI Accepts, published online ahead of print on 12 May 2010

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ABSTRACT 25

We investigated HIV-1-vaccine-induced lymphoproliferative responses in healthy volunteers 26

immunized intradermally or intramuscularly (with or without adjuvant GM-CSF protein) with 27

DNA expressing HIV-1 gag, env, rev and rt at months 0, 1 and 3 using a Biojector and 28

boosted at nine months with MVA expressing heterologous HIV-1 gag, env and pol. 29

Lymphoproliferative responses to AT-2 inactivated HIV-1 antigen were tested by a 3H-30

thymidine uptake assay and a Flow-cytometric Assay of Specific Cell-mediated Immune-31

response in Activated whole blood (FASCIA-WB) two weeks after the HIV-MVA boost 32

(n=38). A FASCIA using peripheral blood mononuclear cells (FASCIA-PBMC) was also 33

employed (n=14). Thirty-five of 38 (92%) vaccinees were reactive by the 3H-thymidine 34

uptake assay. Thirty-two of 38 (84%) vaccinees were reactive by the CD4+ T cell FASCIA-35

WB and seven of 38 (18%) also exhibited CD8+ T cell responses. There was strong 36

correlation between the proliferative responses measured by the 3H-thymidine uptake assay 37

and CD4+ T cell FASCIA-WB (r=0.68; p<0.01). Fourteen vaccinees were analyzed using all 38

three assays. Ten of 14 (71%) and 11/14 (79%) demonstrated CD4+ T cell responses in 39

FASCIA-WB and FASCIA-PBMC, respectively. CD8+ T cell reactivity was observed in 3/14 40

(21%) and 7/14 (50%) using the FASCIA-WB and FASCIA-PBMC, respectively. All 41

fourteen were reactive using the 3H-thymidine uptake assay. The overall HIV-specific T cell 42

proliferative response in the vaccinees employing any of the assays was 100% (38/38). A 43

standardized FASCIA-PBMC, which allows simultaneous phenotyping may be an option to 44

the 3H-thymidine uptake assay for assessment of vaccine-induced T cell proliferation, 45

especially in isotope-restricted settings. 46

47

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INTRODUCTION 48

Human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome 49

(AIDS) is a global burden, but predominantly prevalent in sub Saharan Africa. In 2008 there 50

were an estimated 33.4 million (31.1 – 35.8 million) people living globally with HIV, and an 51

estimated 2.7 million (2.4 – 3.0 million) new HIV infections and 2.0 million (1.7 – 2.4 52

million) HIV related deaths occurred (24). Sixty-seven percent of all people living with HIV 53

are found in sub-Saharan Africa, and 72% of total global AIDS deaths occurred in the region 54

in 2008. Approximately 60% of HIV infection in Sub Saharan Africa is among women (24). 55

Development of an effective and well-tolerated HIV vaccine is likely to be the best way to 56

stop further spread of HIV infection. Most current HIV vaccine candidates in clinical trials are 57

designed to induce cell-mediated immune responses and thus much of vaccine assessment 58

relies on cell-based assays that quantify and characterize the vaccine-induced T cell 59

responses. 60

Several studies provide support for a correlation between T cell function and 61

HIV control (Reviewed in 1). HIV-specific CD4+ T cell responses including vigorous 62

proliferation, production of IFN-γ and β chemokines were shown to be associated with 63

control of virus replication and prevention of disease progression by Rosenberg and 64

colleagues (18). Proliferative capacity has generally been linked to CD4+ T cell responses. 65

However, Migueles et al. reported that CD8+ T cells from HIV-infected long-term 66

nonprogressors (LTNP) had a greater capacity to proliferate than those from progressors. 67

Proliferation was tightly coupled to increases in perforin expression indicating enhanced 68

effector function (17). More recently, CD8+ T cells with proliferative capacity have been 69

implicated in the immune control of HIV-1 infection. HLA-B*57- and B*27- restricted CD8+ 70

T cells from long-term non-progressors exhibited greater proliferative capacity than those 71

restricted by other alleles (13). In a South African cohort of HIV-infected individuals, 72

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proliferation showed a significant inverse correlation with viral load, while cytokine 73

production and degranulation did not (5). Calarota et al (2) recently also showed that the 74

presence of antigen-specific T cell precursors with high proliferative capacity was associated 75

with HIV-1 control and preservation of CD4 counts. Additionally, T cell proliferation has 76

been associated with reduced HIV acquisition in Kenyan female sex workers (12). 77

There are various assays that are used for monitoring of vaccine-induced cell-78

mediated immune responses, of which detection of proliferative capacity is one. The 79

lymphocyte proliferation assay (LPA) has been used for many years to measure in vitro HIV 80

specific T lymphocyte responses (3, 6, 25, 26, 27). LPA results are influenced by antigen 81

processing and presentation, the ability of T cells to proliferate in culture and the initial 82

frequency of antigen-specific memory T cells (9, 14, 25). The tritiated thymidine (3H-83

thymidine) uptake assay is the conventional method that has been used to determine 84

proliferation of T lymphocytes by measurement of incorporation of a radioactive tracer 85

tritiated thymidine in newly synthesized DNA of dividing cells. The assay utilizes purified 86

peripheral blood mononuclear cells (PBMC) and takes 3 to 7 days to complete. The assay 87

does not provide information on the phenotype of proliferating cells and has generally been 88

considered a CD4+ T-cell assay (4). Furthermore, the use of radioactive thymidine is restricted 89

in some developing countries. 90

Several flow cytometry-based methods have been described to measure 91

proliferative T cell responses including the detection of intracellular markers of cell division 92

such as Ki67 (22), incorporation of the thymidine-analogue bromodeoxyuridine (BrdU) (16) 93

and staining with fluorescent dyes such as carboxyfluorescein diacetate, succinimidyl ester 94

(CFSE) and PKH26 (7, 10, 15). CFSE or PKH26 is divided between daughter cells upon cell 95

division whereby the fluorescence intensity of the cells reveals the number of divisions that 96

have occurred. Another LPA called Flow cytometric Assay of Specific Cell-mediated 97

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Immune response in Activated whole blood (FASCIA-WB) utilizes whole blood samples that 98

are cultivated for 6-7 days in the presence or absence of stimulators and results are assessed as 99

the number of antigen-specific lymphoblasts generated (8, 23). The assay can also be run 100

using PBMCs (FASCIA-PBMC). Both FASCIA variants can differentiate between CD4+ and 101

CD8+ T lymphocyte immune responses. In settings where there is restricted use of radioactive 102

thymidine, FASCIA-WB or FASCIA-PBMC may be an option to a conventional 3H-103

thymidine uptake assay. 104

A randomized, open label, phase I HIV safety and immunogenicity study 105

(HIVIS01/02) to assess different modes of administering an HIV DNA vaccine candidate 106

(plasmid DNA with inserted HIV genes env, rev, gag and RT) boosted with heterologous 107

HIV-1 recombinant modified vaccinia virus Ankara (MVA) with analogous genes was 108

completed recently in Stockholm, Sweden. In the HIVIS01/02 trial, 34 of 38 vaccinees had 109

HIV-specific IFN-γ ELISpot responses and 35 of 38 vaccinees had a positive 110

lymphoproliferative response by the 3H-thymidine uptake assay. An overall total of 37 (97%) 111

were responders. It was also shown that a low dose of HIV-1 DNA administered ID was as 112

effective as a high dose IM in priming for the MVA boosting vaccine (21). 113

The aim of the current study was to further define the HIV-1 specific 114

lymphoproliferative responses in these volunteers by applying a flow cytometry based assay 115

employing either whole blood (FASCIA-WB) or PBMCs (FASCIA-PBMC) to assess 116

vaccine-induced CD4+ and CD8+ T cell proliferation. Furthermore, the use of FASCIA, 117

especially suitable in isotope-restricted settings, was explored as an alternative to the 118

conventional 3H-thymidine uptake assay. 119

120

MATERIALS AND METHODS 121

Study samples 122

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Samples from 38 healthy volunteers participating in the HIVIS01/02 trial were 123

included in the study. The immunization scheme and vaccine components used in the 124

HIVIS01/02 trial have been described previously (21). In brief, volunteers were randomized 125

to 4 different treatment arms and immunized either ID or IM, with or without adjuvant GM-126

CSF (Table 1), with DNA expressing HIV-1 genes (subtype) env(A,B,C), rev(B) gag(A,B), 127

and rt(B) at months 0, 1 and 3 using a Biojector (Kindly provided by R. Stout, Bioject, 128

Portland, Oregon). The volunteers were further re-randomized for boosting at nine months 129

with HIV-1 MVA expressing HIV-1 env (E), gag (A) and pol (A) genes developed by 130

Laboratory of Viral Diseases, National Institute of allergy and Infectious Diseases, National 131

Institutes of Health, Bethesda, MD, USA and produced by Walther Reed Army Institute for 132

Research, Rockville, MD, USA (Table 1). 133

Lymphoproliferative responses to AT-2 inactivated HIV-1 antigen were 134

determined in fresh blood samples collected from 38 vaccinees two weeks after the HIV-1 135

MVA boosting immunization using the 3H-thymidine uptake assay and the FASCIA-WB. A 136

FASCIA using purified peripheral blood mononuclear cells (FASCIA-PBMC) was employed 137

during the later part of the study (n=14). The fourteen samples were selected on the basis of 138

their availability, as all assays were performed using fresh cells. 139

Twenty-eight healthy blood donors were also included to determine background 140

reactivity levels, 17 for the FASCIA-PBMC and 27 for the conventional 3H-thymidine uptake 141

assay. 142

143

Antigens 144

Aldrithiol-2 (AT-2) treated HIV-1 MN (subtype B) and SUPT1 microvesicles 145

(control), kindly donated by Dr. J Lifson (National Cancer Institute, Frederick, USA) were 146

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used at a final concentration of 2,5 µg/mL. Phytohemaagglutinin (PHA, 5 µg/mL, Difco 147

Laboratories, Detroit, USA) was used as positive control. 148

149

Peripheral blood mononuclear cells (PBMC) preparation 150

Whole blood was collected in cell preparation tubes (CPT, Becton Dickinson 151

Vacutainer Systems, Franklin Lakes, NJ, USA) containing sodium heparin as anticoagulant 152

and a cell separation medium. The CPTs were processed according to the manufacturer’s 153

instructions. After blood draw, the CPTs were transported to the laboratory, where the tubes 154

were inverted 8 times before centrifugation 1500 x g at room temperature. Cell preparation 155

was performed within 6 hours of blood collection. The cell suspension was collected and the 156

cells were washed twice in PBS and resuspended in complete medium consisting of RPMI 157

1640 medium with Glutamax (Invitrogen Ltd, Paisley, UK), supplemented with penicillin (50 158

IU/mL)-streptomycin (50 µg/mL) and 10 mM HEPES, before counting. PBMC yield and 159

viability were determined using a NucleoCounter (ChemoMetec A/S, Allerod, Denmark). 160

Fresh PBMCs were used in the 3H-thymidine uptake assay and FASCIA-PBMC.

161

3H-thymidine uptake assay 162

The 3H-thymidine uptake proliferation assay was performed as previously 163

described (21). Briefly, fresh PBMCs were incubated in triplicate using 200000 cells/well in 164

96-well flat-bottomed plates (Nunclon, Aahus, Denmark) in 0.2 ml complete medium 165

supplemented with 10% heat inactivated human AB+ sera. Cells were cultured at 37°C in a 166

humidified 7.5% CO2 incubator in the absence (complete medium only) or presence of 167

Aldrithiol-2 (AT-2) treated HIV-1 MN antigen, SUPT1 microvesicles (control antigen) and 168

PHA. Cell cultures were pulsed for 6 hours on day 2 (PHA) and day 6 (specific antigens) with 169

1µCi [3H]-thymidine per well (GE-Healthcare Bio-Sciences AB, Uppsala, Sweden). The cells 170

were harvested onto filters using Harvester 96 MACH III Tomtec (Wallac, Turku, Finland) 171

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and 3H-thymidine incorporation was measured (cpm, counts per minute) using a 1450 172

MicroBeta liquid scintillation counter (Wallac, Turku, Finland). T cell proliferation was 173

reported as stimulation index (S.I.) determined by dividing the mean counts per minute of the 174

antigen-stimulated wells by the mean of the unstimulated control wells. The mean background 175

reactivity in samples from healthy blood donors (n=27) was 1.70±2.09 SI. A SI >8 was 176

considered positive (SI mean +3SD=7.95). 177

178

FASCIA-WB 179

Blood was collected in Vacutainer tubes (BD) containing sodium heparin as 180

anticoagulant. Whole blood diluted 1/8 in medium (RPMI1640 w/glutamax supplemented 181

with 50 IU/ml penicillin and 50 µg/ml streptomycin) was cultured in the absence (medium 182

only, Negative control, NC) or presence of HIV-1 antigen (Test) and PHA (Positive Control, 183

PC). Four hundred µL of the diluted blood and 100 µL of antigen or medium only were added 184

to polystyrene round-bottomed tubes with caps (BD Biosciences, San Jose, CA, USA) and 185

incubated for 7 days in a humidified atmosphere at 37°C with 7.5% CO2. All samples were 186

run in duplicates. After incubation, the tubes were centrifuged at 300 x g for 5 minutes and 187

350 µL of the supernatants were removed. The cells were stained with anti-CD3 FITC and 188

anti-CD4 PerCP (BD) for 15 minutes in the dark at room temperature. One mL of lysing 189

solution, (Pharm Lyse, BD) was added and incubated for 10 minutes at room temperature, 190

followed by centrifugation at 300 x g for 5 minutes, removal of the supernatants, addition of 191

lysing solution (BD), washing with FACSFlow (BD), and resuspension in 450 µL of Cell fix 192

(BD). The samples were stored in the dark at 4 °C until acquisition. 193

194

FASCIA-PBMC 195

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Fresh PBMCs were cultured in complete medium supplemented with 10% heat 196

inactivated human AB+ sera in the absence (complete medium, negative control, NC) or 197

presence of HIV-1 antigen (Test) and PHA (Positive control, PC). Two hundred thousand 198

cells/well (100 µL) and 100 µL of antigen or complete medium were added to 96-well flat-199

bottomed plates (Nunclon, Aahus, Denmark) and incubated for 7 days in a humidified 200

atmosphere at 37°C with 7.5% CO2 in air. All samples and controls were run in duplicates. 201

After stimulation, the cells were transferred to polystyrene round-bottomed tubes (BD), 202

centrifuged at 300 x g for 5 minutes and 100 µL of the supernatants were removed. The 203

pellets were stained with anti-CD3 FITC and anti-CD4 PerCP (BD) for 10 minutes at room 204

temperature, followed by washing with FACSFlow (BD), and resuspension in 450 µL of Cell 205

fix (BD). The samples were stored in the dark at 4°C until acquisition. 206

207

Flow cytometric acquisition 208

Acquisition was performed using either a FACSCan (BD) equipped with one 15 209

mW air argon-ion (488 nm) or a FACSCalibur (BD) equipped with one 15 mW air argon-ion 210

and one red iodide laser operating at 488 nm and 635 nm, respectively. The instrument was 211

set for three or four-colour analysis using FACSComp software (BD) in conjuction with 212

Calibrite beads (BD), with a threshold on forward scatter (FSC) to exclude debris. The flow 213

rate was set on High which corresponds to a collection rate of 1 µL/sec. Further, the collection 214

criterion was set on time instead of events in order to collect one tenth (45 µL) of the sample 215

volume from each tube, which enables determination of the total number of the cell subsets in 216

a culture by multiplying the number of acquired cells with a factor of 10. Data were collected 217

in list mode and analyses were performed using Cell Quest Pro software version 4.0.1 and 218

FlowJo software version 8.7.1. 219

220

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Flow cytometric analysis and calculations 221

A detailed description of the sample analysis is found in Figure 1. All samples 222

were analyzed as duplicates. Proliferative responses were measured by detection of 223

CD3+CD4+and CD3+CD4- (CD8+) lymphoblasts and results were expressed as mean 224

percentage stimulation (%S): [100 x (Test-NC) / (PC-NC)]. For simplicity, CD3+CD4+ and 225

CD3+CD4- T cells will be referred to as CD4+ and CD8+ T cells, respectively. A value above 226

the mean ± 3SD %S of the baseline values of the vaccinees was considered to be a positive 227

reaction for FASCIA-WB. The mean background reactivity levels in the baseline samples 228

from the vaccinees (n=38) were 0.23±0.24 %S for the CD4+ T cells and 0.21±0.28 %S for the 229

CD8+s T cells. A %S above 1.0 and 1.1, respectively, was considered to be a positive 230

response. 231

In the absence of baseline values for FASCIA-PBMC, values above the mean ± 232

3SD %S of 17 healthy normal controls was considered positive in this assay. The mean 233

background reactivity levels were 0.26±0.72 %S for the CD4+ T cells and 0.55±0.75 %S for 234

the CD8+ T cells. A %S >2.4 and >2.8, respectively, was considered to be a positive response. 235

236

Ethical considerations 237

The Phase I HIVIS trial protocols were approved by the Regional Ethics 238

Committee, Stockholm, Sweden. Informed consents were obtained from study volunteers 239

prior to their inclusion into the study. 240

241

Statistical analysis 242

The proportion of vaccinees with HIV-1 specific lymphoproliferative responses 243

was determined for each assay (3H-thymidine uptake, FASCIA-WB and FASCIA-PBMC 244

assay) and comparisons were made using Pearsons Chi-square-test and Fisher’s exact test, 245

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where applicable. The Mann-Whitney U test was used for comparative analyses of LPA 246

reactivity levels. Correlations between results obtained by the 3H-thymidine uptake assay, 247

FASCIA WB and FASCIA PBMC were estimated by non-parametric Spearman´s Rank 248

correlation test. A p-value of <0.05 was considered statistically significant. 249

250

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RESULTS 251

The 3H-thymidine uptake assay displayed strong T cell proliferative responses 252

Lymphoproliferative responses to AT-2 inactivated HIV-1 antigen were 253

determined in fresh blood samples collected from 38 vaccinees two weeks after the HIV-1 254

MVA boosting immunization using a 3H-thymidine uptake assay and FASCIA-WB. Thirty-255

five of 38 vaccinees (92.1%) were reactive by the 3H-thymidine-uptake assay (SI>8) with a 256

mean±SD reactivity of 135.63±233.10 SI (Fig. 2). The mean±SD counts per minute in 257

reactive donors was 17987±18954 (range 386-72295). 258

259

Both HIV-1-specific CD4+ and CD8+ T cell proliferative responses were detected by 260

FASCIA-WB 261

Thirty-three of 38 vaccinees (86.8%) were reactive by the CD4+ T cell FASCIA-262

WB (Fig. 3a) and 8 (21%) were also reactive by the CD8+ T cell FASCIA-WB (Fig. 3b). The 263

mean (±SD) HIV-specific CD4+ T cell proliferative responses detected by the FASCIA-WB 264

was 0.23±0.24 %S in the baseline samples and 10.42±16.26 %S in the HIV-1 MVA post-265

vaccination samples. The corresponding values for the CD8+ T cell proliferative responses 266

were 0.21±0.28 and 3.21±10.97 %S, respectively. 267

268

The FASCIA-PBMC (CD4+) assay correlated with the 3H-thymidine uptake assay 269

A slightly higher number of responders (35/38) were detected by the 270

conventional 3H-thymidine uptake assay compared to FASCIA-WB (33/38) without reaching 271

statistical significance. Five vaccinees that were reactive by the 3H-thymidine uptake assay 272

were non reactive by the CD4+ T cell FASCIA-WB and three vaccinees that were reactive by 273

the CD4+ T cell FASCIA-WB were non reactive by the 3H-thymidine uptake assay (Fig. 4). 274

Six of the eight samples with discordant results displayed reactivities close to the cut-off in 275

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both assays. Nonetheless, there was a significant correlation (r=0.68; p<0.01) between the 276

proliferative responses measured by the 3H-thymidine uptake assay and the CD4+ T cell 277

FASCIA-WB. 278

279

The FASCIA-PBMC assay reveals additional T cell reactivity 280

Because of the discrepancy in the number of responders noted using FASCIA-281

WB compared to the 3H-thymidine uptake assay, a FASCIA using PBMC was added for the 282

testing of the final 14 vaccinees in the trial. All fourteen (100%) were reactive using the 3H-283

thymidine uptake assay, eleven (78.6%) were reactive by the CD4+ T cell FASCIA-PBMC 284

and ten (71.4%) by the CD4+ T cell FASCIA-WB (p=0.78 and p=0.59, respectively) (Fig. 5). 285

The mean (±SD) HIV-specific CD4+ T cell proliferative responses detected by the FASCIA-286

PBMC and FASCIA-WB were 32.82±47.64 %S and 14.44±24.29 %S, respectively (p=0.11). 287

There was a significant correlation in CD4+ T cell proliferative responses (r=0.90, p<0.001) 288

between FASCIA-WB and FASCIA-PBMC. Seven (50%) were reactive by the CD8+ T cell 289

FASCIA-PBMC and 4 (28.6%) by the CD8+ T-cell FASCIA-WB (p=0.13). The mean (±SD) 290

HIV-specific CD8+ T cell proliferative responses detected by the FASCIA-PBMC and 291

FASCIA-WB were 9.53 ±13.58 %S and 7.50±17.57 %S, respectively (p=0.49). 292

293

T cell proliferative responses by vaccination group 294

The overall HIV-specific T cell proliferative response in the vaccinees 295

employing any of the three assays was 100% (38/38). However, since only four or five 296

volunteers were included in each vaccination group (A1-D2) no attempt was made to compare 297

T cell proliferative responses by HIV-DNA immunization and HIV-MVA dose. We compared 298

intramuscular (n=19) and intradermal (n=19) delivery of the HIV-MVA vaccine regardless of 299

dose and HIV-DNA priming. There was a borderline significance in reactivity by route of 300

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HIV-MVA immunization observed using the conventional 3H-thymidine uptake assay, 301

p=0.0597 or the CD4+ T cell FASCIA-WB, p=0.0597 (Fig. 6). 302

303

DISCUSSION 304

While the CD8+ T cells provide the effector arm of the cell mediated immune 305

response, the CD4+ T-cells influence their maturation and function and an efficacious cell 306

based HIV vaccine will most likely have to induce both CD4+ and CD8+ T cell responses. 307

Additionally, HIV-specific CD4+ T cells should be capable of proliferating and expanding 308

after antigen exposure in order to preserve the function of recall HIV-specific CD8+ T cells. 309

In the current study, HIV-1 vaccine-induced lymphoproliferative responses were determined 310

by using a conventional 3H-thymidine uptake assay and two flow-cytometric assays, the 311

FASCIA-WB and the FASCIA-PBMC. The latter two assays allow further characterisation of 312

the proliferating cells by immunophenotyping. We found that the 3H-thymidine uptake assay 313

detected a slightly higher number of responders (35/38) compared to FASCIA-WB (33/38). 314

We also detected a higher number of responders with HIV-specific CD8+ T cell proliferative 315

responses by FASCIA-PBMC as compared to FASCIA-WB (8/14 vs. 4/14). Overall, T cell 316

proliferative responses were frequent and seen in both CD4+ T cell and CD8+ T cell 317

compartments. 318

Our findings extend those reported by others testing DNA prime-poxvirus 319

vector vaccine candidates in clinical trials. Goonetilleke and colleagues reported induction of 320

HIV-1 specific T cells with proliferating capacity using 3H-thymidine incorporation in 5 of 8 321

volunteers receiving vaccines vectored by plasmid DNA and modified vaccinia Ankara 322

expressing HIV-1 24/p17 gag linked to a string of CD8+ T cell epitopes (10). Using CFSE 323

labeling, a LPA that also allows detection of CD4+ and CD8+ T cells, the induction of CD4+ 324

and CD8+ T cell proliferation in response to Env and Gag-specific stimulation was recently 325

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shown in three vaccinees receiving a recombinant DNA prime and boosting with the poxvirus 326

vector NYVAC expressing Env, Gag, Pol and Nef polypeptide domain from HIV-1 (11). 327

This is the first report where FASCIA has been used for the assessment of 328

vaccine-induced T cell proliferative responses. In the current study, AT-2 treated HIV-1 MN 329

antigen (subtype B) was used in the T cell proliferation assays and stimulation of both CD4+ 330

and CD8+ T cells were detected, although CD4+ T cells were more frequent. AT-2 treatment 331

results in elimination of infectivity with preservation of conformational and functional 332

integrity of virion surface proteins including both virally encoded determinants and proteins 333

derived from the host cells in which the virus was produced (19). In a recent study of 18 US 334

individuals infected with HIV-1 subtype B and 32 Ugandan individuals infected with 335

subtypes A and D or recombinants AC and AD, both subtype-specific and cross-reactive 336

responses were observed. Furthermore, as also shown here, AT-2 inactivated HIV-1 antigen 337

was reported to stimulate both CD4+ and CD8+ HIV-1-specific responses (20). 338

We detected a slightly higher number of responders in the 3H-thymidine uptake 339

assay compared to FASCIA-WB (35/38 vs. 33/38). These findings suggested that the 340

conventional 3H-thymidine uptake assay might be more sensitive compared to FASCIA-WB. 341

In a study by Svahn and colleagues the FASCIA-WB displayed a sensitivity of 95% for the 342

detection of varizella-zoster virus–specific cell-mediated immune responses in VZV-antibody 343

positive children with a history of chicken-pox (23). We found a higher number of responders 344

with HIV-specific CD8+ T cell proliferative responses when using purified PBMC in the 345

FASCIA, compared to using whole blood FASCIA (8/14 vs. 4/14). The FASCIA-PBMC is 346

similar to the conventional 3H thymidine uptake assay in that a defined number of cells 347

(200000/well) are activated. In the whole blood assay (FASCIA-WB) the number of cells that 348

are activated varies depending on the blood counts. Furthermore, in the FASCIA-WB assay 349

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all blood components, including antibodies are present. Thus, the proliferating cells might be 350

affected by the 7-day whole blood culture environment. 351

The 3H-thymidine uptake assay is based on the incorporation of a radioactive 352

tritiated thymidine in newly synthesized DNA of dividing cells, while the FASCIA-WB and 353

FASCIA-PBMC assays are based on the assessment of the number of antigen-specific 354

lymphoblasts generated during the proliferation procedure. A main limitation of the 3H-355

thymidine uptake assay is that it only measures the total amount of DNA-synthesis in bulk 356

culture and does not allow phenotyping of proliferating cells. FASCIA-WB is less laborious 357

and time-consuming, suitable for large-scale studies, it does not require the use of radioactive 358

substance and is advantageous over the 3H-thymidine uptake assay since it allows 359

simultaneous phenotyping of the responsive T cells identifying both CD4+ and CD8+ T cells. 360

It can also be combined with characterization of the cytokine profile of responding cells using 361

intracellular staining or examination of culture supernatants. However, formation of 362

microcoagel in the sample collection tube or in the culture tube might affect the final result of 363

the assay. This applies for any whole blood assay. 364

Utilization of a defined number of peripheral blood mononuclear cells (200000 365

cells/well) and standardized culture conditions, offers a greater possibility of assay 366

standardization including a reduced risk of reduced sensitivity due to the use of samples from 367

lymphopenic individuals. In the FASCIA-WB assay, the number of PBMC used is not 368

standardized, and antibodies and other components that might inhibit the proliferation 369

procedure are present in the culture. The FASCIA-PBMC has the combined advantages of the 370

3H-thymidine uptake assay and the FASCIA-WB: standardized number of PBMC and culture 371

conditions; allows further characterisation of the responsive T cells by simultaneous 372

phenotyping, intracellular cytokine staining or analysis of culture supernatant; no use of 373

radioactive substance and it is potentially suitable for large-scale studies. Further evaluations 374

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of the FASCIA-PBMC assay in clinical trial settings are needed and a comparison of the 375

FASCIA-PBMC assay and CFSE labelling assay is ongoing in our laboratory. 376

In conclusion, strong HIV-1 specific CD4+ and CD8+ T cell lymphoproliferative 377

responses were demonstrated in a high proportion of volunteers in the HIVIS01/02 study 378

following HIV-1 DNA/MVA immunization. A standardized FASCIA-PBMC assay, which 379

allows simultaneous phenotyping, may be an option to the conventional 3H-thymidine uptake 380

assay for assessment of vaccine-induced T cell proliferation, especially in isotope-restricted 381

settings. 382

383

ACKNOWLEDGMENTS 384

We extend our special thanks to the study volunteers. We also thank Bo Hejdeman for serving 385

as the responsible study physician for the HIVIS trial; Andreas Bråve for participating in the 386

development of the HIV DNA vaccine; Bernard Moss and Patricia Earl for design, 387

construction and characterization of the HIV MVA vector; Deborah Birx for the intellectual 388

design, GMP production and QA/QC of the MVA vaccine; Tom Van Cott for design, 389

contruction, cGMP manufacturing, release testing and safety/toxicity studies as well as 390

preclinical immunogenicity testing of the MVA; Josephine Cox for participation in the study 391

design of the HIVIS trial, standardization of SOPs and peptides; Nelson Michael and Merlin 392

Robb for MVA vaccine production and HIVIS trial protocol development. This work was 393

supported by funding from the Swedish International Development Cooperation Agency 394

(Sida), Department for Research Cooperation, SAREC (SWE-2004-120 to CN, HIV2004-395

000809 to GB, 2004:813 to GB) and the European Union (INCO-DEV A4 ICFP501A4PR03 396

to ES). 397

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505 506

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FIGURE LEGENDS 507

1. Analysis strategy for flow cytometric assessment of lymphoproliferative responses by 508

detection of lymphoblasts in FASCIA-WB or FASCIA-PBMC. Proliferative responses 509

against AT-2 inactivated HIV-1 antigen assessed by FASCIA-WB in samples from a vaccinee 510

collected at baseline (column A) and two weeks after MVA boost (column B) and by 511

FASCIA-PBMC in a sample from a blood donor (column C) and from a vaccinee at two 512

weeks after MVA boost (column D). CD3+ T cells are identified by region R1, on a side 513

scatter (SSC) vs. FL1 (CD3) dot plot (upper row). Then, small nongranular resting 514

lymphocytes and granular lymphoblasts are identified by region R2 and R3, respectively, on a 515

forward scatter (FSC) vs. side scatter (SSC) dot plot (second row). Cells within R2 and R3, 516

are displayed on dot plots showing staining with anti-CD3 and anti-CD4 antibodies, where 517

CD4+CD3+ T cells and CD4-CD3+ T cells are indicated as CD4 and CD8, respectively. 518

519

2. T cell proliferation against AT-2 inactivated HIV-1 antigen measured by the 3H-thymidine 520

uptake assay (cut-off ≥ 8 SI) in 38 HIVIS01/02 volunteers, at time of HIV-MVA vaccination 521

(grey bars) and two weeks after vaccination (black bars) by vaccination group. The 522

vaccination groups are detailed in Table 1. 523

524

3. a) CD4+ (cut-off >1.0 %S reactivity); b) CD8+ (cut-off >1.1 %S reactivity) T cell 525

proliferation assessed by FASCIA-WB in 38 HIVIS01/02 volunteers, following stimulation 526

with AT-2 inactivated HIV-1 antigen, at baseline (grey bars) and two weeks after HIV-MVA 527

vaccination (black bars). The vaccination groups are detailed in Table 1. 528

529

530

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4. Correlation of T cell proliferative responses against AT-2 inactivated HIV-1 antigen 531

measured by the 3H-thymidine uptake assay and the FASCIA-WB (CD4+) in 38 HIVIS01/02 532

volunteers 2 weeks after the HIV-MVA immunization (r=0.68; p<0.01). Samples reactive in 533

both assays (blue rhombs), reactive only in the 3H-thymidine uptake assay (red triangles) and 534

only FASCIA-WB (CD4+, yellow circles) are indicated. 535

536

5. CD4+ and CD8+ T cell proliferation assessed by FASCIA-WB (yellow circles) and 537

FASCIA-PBMC (black triangles) in 14 volunteers, against AT-2 inactivated HIV-1 antigen. 538

A %S above 1.0 and 1.1 for CD4+ and CD8+ T cells, respectively, was considered to be a 539

positive response in the FASCIA-WB assay. A %S above 2.4 and 2.8 for CD4+ and CD8+ T 540

cells, respectively, was considered to be a positive response in the FASCIA-PMBC assay. 541

There is less variability in CD4+ than in CD8+ T cell proliferation between FASCIA-WB and 542

FASCIA-PBMC. 543

544

6. T cell proliferative responses by route of HIV-MVA immunization determined by the 545

conventional 3H-thymidine uptake assay (A) and CD4+ T cell FASCIA-WB (B). 546

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547 Table 1. 548 Immunization

group 3x HIV DNA priming

immunizations Immunization

group 1 x HIV MVA boosting

immunization

A (n=10) 1 mg ID A1 (n=5) 108 PFU IM A2 (n=5) 107 PFU ID

B (n=10) 3.8 mg IM B1 (n=5) 108 PFU IM B2 (n=5) 107 PFU ID

C (n=9)* 1mg + GM-CSF ID C1 (n=5) 108 PFU IM C2 (n=4)* 107 PFU ID

D (n=9)* 2 mg + GM-CSF IM D1 (n=4)* 108 PFU IM D2 (n=5) 107 PFU ID

HIV DNA priming immunizations were administered at days 0, 30 and 90. The HIV MVA 549

boosting immunization was administered at month 9.. GM-CSF, granulocyte 550

macrophage colony stimulating factor; ID, intradermal; IM, intramuscular 551

*Two vaccinees did not receive all immunizations and were therefore not included in the analyses. 552

553 554

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Figure 1. 555 556

CD3 CD3

CD3

CD3+ T-lymphocytes

(R1)

R2

R3

R2

R3R3

R2R2

R3

FASCIA-WB FASCIA-PBMC

ABaseline

CControl sample

CD3

CD3

Scatter plot(R2 and R3)

RestingLymphocytes

(R2)

Lymphoblasts(R3)

D2 w post-MVA

CD4

CD4

CD8

CD4

CD8

CD4

CD8

CD4

CD8

CD4

CD8

CD4

CD8

CD4

CD8

CD4

CD8

B2 w post-MVA

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Figure 2. 557

0

50

100

150

200

250

300

350

400

At the time of HIV-MVA vaccination 2 weeks after HIV-MVA vaccination

A1 C2C1B2B1A2 D1 D2

Vaccination group

1385T

ce

ll p

roli

fera

tio

n (

S.I

.)

558

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Figure 3 559

CD

4+

T c

ell

pro

life

rati

on

(%S

)

0

5

10

15

20

25

30

35

40

Baseline 2 weeks after HIV-MVA vaccination

A1 D1C2A2 B1 B2 C1 D2

88

0

1

2

3

4

5

6

7

8

9

10

CD

8+

T c

ell

pro

life

rati

on

(%

S)

Baseline 2 weeks after HIV-MVA vaccination

A1 D2D1C2C1B2B1A2

43.7 53.6

Vaccination group

A

B

560

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561 Figure 4. 562 563

0

50

100

150

200

250

300

350

400

0 4 8 12 16 20 24 28 32 36 403H

th

ym

idin

e i

nc

orp

ora

tio

n (

S.I.)

FASCIA-WB CD4 (%S)

564

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Figure 5. 565 566

0

0

1

10

100

1000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14

CD4 CD8

T c

ell p

rolife

rati

on

(%

S)

Volunteer

Whole Blood PBMC

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Figure 6. 567 568

p=0.0597 p=0.0597

569

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