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: [email protected] 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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