Conceived and designed the experiments: MR. Performed the experiments: PV MA VGW EJ MPJ JB AG. Analyzed the data: PV MA VGW MPJ JL AMS JB AG MR. Contributed reagents/materials/analysis tools: MPJ JL AMS JB AG MR. Wrote the paper: PV JL MR.
Andres M. Salazar is Chief Executive Officer and Scientific Director of Oncovir Inc., which provided PolyICLC (Hiltonol). This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
HIV-infected individuals rely on antiretroviral therapy (ART) to control viral replication. Despite abundant demonstrable benefits, the multiple limitations of ART point to the potential advantages of therapeutic vaccination approaches that could provide sustained host control of viral replication after discontinuation of ART. We provide evidence from a non-human primate model that a therapeutic vaccine applied to the tonsils can maintain low viral loads after cessation of ART.
Animals received 40 weeks of ART initiated 9 weeks after rectal SIVmac239 infection. During ART, animals were vaccinated (or not) with AT-2 inactivated SIVmac239 using CpG-C ISS-ODN (C274) or polyICLC as adjuvants. PolyICLC/AT-2 SIV vaccinated animals maintained viral loads <3×103 copies/ml for up to 16 weeks post-ART, whereas the C274/AT-2 SIV vaccinated and non-vaccinated animals' viremia ranged between 1×104–4×105 copies/ml (p<0.03). Neutralizing Ab activity in plasma was increased by polyICLC/AT-2 tonsillar vaccination under ART, compared to controls (p<0.03). Subsequent vaccination of all animals with polyICLC/AT-2 SIV in the absence of ART did not alter viral loads. Other immune parameters measured in blood and tissues were comparable between groups.
These results provide support for the potential benefit of mucosally delivered vaccines in therapeutic immunization strategies for control of AIDS virus infection.
Human immunodeficiency virus (HIV-1) infection affects more than 33 million people world-wide, including more than 300,000 children (WHO/UNAIDS). Left untreated, it leads to acquired immune deficiency syndrome (AIDS) and death. More than 2 million people a year die of AIDS-related illness. Whilst a vaccine for HIV remains elusive, major advances in therapies have been made in the last decade.
Highly active anti-retroviral therapy (HAART), introduced in 1996, has so far been very effective in controlling viral replication and preventing the progression of treated patients to AIDS
Therapeutic vaccination offers the promise of enhancing host antiviral immune responses by immunization under the cover of antiretroviral drug suppression of viral replication, potentially enabling durable control of viral replication even after subsequent discontinuation of HAART. However, most studies of therapeutic vaccination to date in HIV infected subjects have been disappointing
Aldrithiol 2 (AT-2) inactivated SIV has been shown to be effective in inducing responses that can help control SIV infection in macaques
Vaccine immunogens are typically administered with adjuvants in order to stimulate more optimal immune responses associated with protective effects. Regularly used adjuvants range from broadly immunostimulatory compounds, such as alum, to bacterial toxins, such as cholera toxin B. Toll-like receptor (TLR) ligands have recently been used as vaccine adjuvants due to their capacity to mimic components of pathogens and stimulate immune responses. CpG-C immunostimulatory oligonucleotides (CpG-C ISS-ODN) bind to the intracellular TLR9 and have been shown to effectively activate plasmacytoid DC (PDC) and B cell responses
Even though a therapeutic vaccine does not seek to prevent establishment of infection after mucosal exposure in the manner a preventative vaccine might, for therapeutic immunization mucosal vaccination has distinct advantages over other routes, such as ease of administration and lower cost. In this study, we tested tonsillar therapeutic vaccination with AT-2 SIVmac239 adjuvanted with CpG-C ISS-ODN vs clinical grade polyIC (Hiltonol or polyICLC). We provide direct evidence of improved virus control under ART after tonsillar immunization with AT-2 SIV and polyICLC. This suggests that non-invasive mucosally applied therapeutic vaccines augmented with polyICLC show promise in controlling AIDS virus replication.
Adult male Chinese Rhesus macaques (
Immunization | Animal ID | ART responsive | IFNγ | SIV Ab responses | CD4 counts | |||||||
Plasma | Rectal | Duodenum | Ileum | Jejunum | Pre | Wk 1 post ART | Wk 26 post ART | Final (necropsy) | ||||
C274/AT-2 SIV | GJ39 | + | + | + | + | + | + | - | 850 | 302 | 683 | 1013 |
GJ47 | + | - | + | + | + | - | + | 944 | 508 | 584 | ||
GJ48 | + | + | + | + | + | - | - | 368 | 842 | |||
GJ50 | + | + | + | + | + | - | - | 482 | 1083 | 283 | ||
GJ53 | + | + | + | + | + | + | - | 991 | 1357 | 1009 | 785 | |
BG93 | + |
+ | + | + | + | + | + | 584 | 538 | 672 | 272 | |
polyICLC/AT-2 SIV | GJ55 | + | + | + | + | + | + | + | 1020 | 192 | 824 | 1092 |
GJ57 | + | + | + | + | + | + | n/a | 1250 | 368 | 708 | 2091 | |
CC48 | + | + | + | + | + | - | + | 390 | 440 | 477 | 235 | |
DV67 | + | + | + | + | + | - | - | 121 | 537 | 457 | 598 | |
DD94 | + | + | + | + | + | - | - | 351 | 394 | 637 | 502 | |
GJ97 | + |
+ | + | + | + | - | - | 499 | 744 | |||
Control | DA47 | + | - | + | + | + | - | - | 796 | 510 | 788 | 637 |
CM96 | + | + | + | + | + | n/a | n/a | 912 | 583 | 479 | 651 |
|
CK25 | + | + | + | + | + | n/a | n/a | 579 | 290 | 458 | 458 |
|
CL68 | + | + | + | + | + | n/a | n/a | 597 | 627 | 361 |
||
EL02 | - | + | + | + | + | n/a | n/a | 608 | 285 | n/a | n/a | |
GJ65 | - | + | + | + | + | n/a | n/a | 751 | 686 | n/a | n/a |
*indicates partial ART responsiveness (i.e. viremia >30 copies/ml; see
Early necropsy due to sickness.
Immune responses were followed by collecting EDTA blood (<10 ml/kg/month) and mucosal (oral and rectal) fluids throughout the study. Mucosal fluids were collected as described previously
CpG-C ISS-ODN C274 was provided by Dynavax Technologies (Berkeley, CA). The sequence was: C274
Macaque peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque density gradient configuration (GE Healthcare, Sweden). Cells were cultured in complete RPMI 1640 (Cellgro, Springfield, NJ) containing 2 mM L-glutamine (GIBCO Life Technologies, Grand Island, NY) 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (GIBCO Life technologies), 50 µM 2-mercaptoethanol (Sigma), penicillin (100 U/ml)/streptomycin (100 µg/ml) (GIBCO Life Technologies) and 1% heparinized human plasma (Innovative Research, Southfield, MI).
Tissue biopsies were placed in RPMI (supplemented as above, but with 10% heat inactivated fetal bovine serum (Mediatech, Manassas, VA) instead of human plasma) containing 200 µg/ml gentamycin (GIBCO) for 1 hour at 4°C. Lymph nodes were cut in 2–4 mm pieces using a forceps and a scalpel and pushed through a 70 µm nylon filter (BD Falcon) with a glass rod. The suspension was spun at 340 g for 10 min. Cells were then resuspended in RPMI (10% FBS) and counted. Jejunum and ileum were washed by spinning at 244 g for 10 min and resuspended in RPMI (10% FBS) containing 0.5 mg/ml Collagenase II (Sigma, St Louis, MO) and 1 mg/ml DNAse I (Roche, Indianapolis, IN) and 1 mg/ml hyaluronidase (Sigma) in a T25 tissue-culture flask (BD Falcon). The tissue was broken up using a forceps and a scalpel and incubated at 37°C for 30 min. The suspension was then passed through a metal sieve using a glass rod. Medium with enzymes, as above, was added to the remaining tissue and incubated at 37°C for a further 30 min. The suspension was passed through a metal sieve again and the filtered suspension from both cases was then filtered using a 70 µm nylon filter. The remaining cell suspension was spun at 340 g for 10 min. Cells were then resuspended in RPMI (1% human plasma) and counted.
Plasma samples were collected from all animals at all time points of the study, as described previously
Neutralizing Ab activity against SIVmac251 was measured in monkey plasma samples, as described previously
SIV-specific IgA was measured in rectal and/or intestinal fluids collected at the beginning of the study, at the last time point prior to vaccination (week 26) and at necropsy by ELISA as previously described
Numbers of IFN-γ spot-forming cells (SFCs) responding to AT-2 SIVmac239 in blood were measured by ELISPOT
Multi-color flow cytometry was used to characterize leukocyte subsets and polyfunctional T cells in macaque blood and tissues. T cells were characterized using Pacific Blue-conjugated anti-CD3 and PerCP-Cy5.5-conjugated anti-CD4 (clones SP34-2 and L200, BD Biosciences), APC-conjugated anti-CD28 (clone CD28.2, BD Biosciences), PE-conjugated anti-CD95 (clone DX2, BD Biosciences), PE-Cy7-conjugated anti-CD25 (clone M-A251, BD Biosciences), PE-conjugated anti-PD-1 (clone J105, eBioscience), PE-Texas Red conjugated anti-CD38 (clone HIT2, Invitrogen) and APC-Cy7-conjugated anti-CD69 (clone FN50, BD Bioscie3nces). Alexa488-conjugated anti-FoxP3 (clone PHC101, eBioscience) was used to identify Tregs. Intracellular cytokine staining (ICS) was performed as previously described
Appropriate irrelevant specificity isotype Ig negative controls were included in all experiments and typically gave MFIs of <1 log. Samples were acquired on a LSR-II (BD) and analyzed using FlowJo software (Tree Star, OR).
RNA was extracted from cell pellets (10×106 cells) using the RNeasy Mini Kit (Qiagen, MD) according to the manufacturer's instructions. RNA was immediately quantified using a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) and converted into cDNA using the SuperScript VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, CA). Primers for IFNα, IFNβ, IFNγ, TNFα, IL-2, IL-6, IL-10, IL-12, CCL4, CCL5, CXCL10, FoxP3, IDO and TGFβ were designed using Primer Express (Applied Biosystems, Foster City, CA) and manufactured by IDT (Coralville, IA). The PCR mixture was set-up as follows, per reaction: 12 µl SYBR Green Master Mix (Applied Biosystems, Warrington, UK), 100 nM forward primer, 100 nM reverse primer, 50 ng cDNA and the reaction was made up to 25 µl with distilled water. The program used was 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The reactions were run on an ABI 7700 machine (Applied Biosystems, CA).
Data were analyzed for statistical significance using the Wilcoxon Rank-Sum test in all cases apart from within-group before-after comparisons for neutralizing Ab titers, where the one-sided Wilcoxon Signed-Rank test was used.
TLR ligands have been previously shown to be effective inducers of the innate immune response and effective adjuvants. We thus set out to examine two different TLR ligands, polyICLC and CpG-C ISS-ODN C274, as adjuvants in therapeutic vaccination with AT-2 inactivated SIVmac239 in SIVmac239 infected Rhesus macaques. Tonsillar vaccination was used as a model of targeting oral mucosal-associated lymphoid tissue and has been shown by us and other laboratories to be an effective port of entry for immunogens
18 SIV-naïve Chinese Rhesus macaques were inoculated rectally with pathogenic SIVmac239. Infection was confirmed by plasma SIV RNA PCR. Statistical analyses of early plasma viremia (weeks 1–9) showed no differences between the groups, including the setpoint viremia at 9 weeks when ART was initiated. 12 animals were vaccinated 4 times, at weeks 26, 32, 38 and 44 post-infection. 6 received the C274/AT-2 SIV vaccine, whereas the other 6 received the polyICLC/AT-2 SIV vaccine. 6 control animals were not vaccinated. 5 weeks after the final vaccination, ART was withdrawn and the animals were followed up for a further 4 months.
All animals reached peak viremia at 2 weeks post-infection (
(A) Plasma SIV RNA copies/ml were determined by PCR. Each symbol indicates an individual animal. The ART treatment period is indicated by the shaded grey box. Arrows indicate immunization time-point. (B) Average (geometric means) viral loads (±SEM) are shown for the ART-responding animals (5 in the C274/AT-2 SIV group, 5 in the polyICLC/AT-2 SIV group and 4 in the control group). Asterisks indicate significant differences between: polyICLC vs both C274 and controls at weeks 2, 4, 8 and 16, p<0.03; polyICLC vs C274 at weeks 3 and 10, p<0.02; polyICLC vs control at week 13, p<0.03. (C) Average Area Under the Curve (AUC) is shown for the ART-responding animals in each group (±SEM). p<0.03 for polyICLC vs C274 and p<0.02 for polyICLC vs control.
Vaccination did not have a demonstrable effect on viral load during the time animals were receiving ART as animals had suppressed plasma viremia to below measurable levels prior to the first immunization (
SIV-specific IFN-γ responses were negligible during ART, increasing post ART in all groups. Comparable levels of SIV-specific T cell responses (normalized by subtracting any background in response to MVs) were detected in the blood of the three treatment groups, by IFN-γ ELISPOT (
(A) Average SIV-specific (to AT-2 SIV) IFNγ ELISPOT responses are illustrated (±SEM). SIV specific responses were determined by subtracting the appropriate MV-induced background. (B) Percentage of cytokine producing (IFNγ, TNFα, IL-2 and combinations) CD3+CD4+ (upper panel) and CD3+CD4− (lower panel) cells were measured in PBMC, by flow cytometry at weeks 17–18 post-ART. Results shown in panels A and B (±SEM) are for 5 animals in the C274/AT-2 SIV group, 5 in the polyICLC/AT-2 SIV group and 4 in the control group.
Neutralizing Abs against SIVmac251 were detected in the blood of all animals post-infection. No differences were observed between animals assigned to the different treatment groups prior to vaccination. Following vaccination with polyICLC/AT-2 SIV under ART, neutralizing Ab titers increased ≥25-fold in all (6 of 6) vaccinees (
Neutralizing Ab titers in the plasma were determined before and after vaccination (vaccination on weeks 26, 32, 38, and 44 post infection). Levels were measured in samples taken 22 weeks after infection and 46 weeks post infection for all animals except CK25 (week 42) and EL02 and GJ65 (week 49) (asterisk). The titer (the last dilution tested at which infection was blocked) is shown for each animal and the mean for each group is marked by the horizontal bar. The circled symbols denote the animals that were ART partial or non-responders. Animal ID numbers are indicated for animals with outlier values relative to the rest of their respective groups.
The major histocompatibility complex (MHC) profile of each animal was determined to ascertain if the animals with the reduced viremia in the polyICLC-treated group had MHC alleles associated with control of viral replication. The following molecules were tested for: Mamu-A*01, Mamu-A*02, Mamu-A*08, Mamu-A*11, Mamu-B*01, Mamu-B*03, Mamu-B*04, Mamu-B*08, Mamu-B*17 and Mamu-DRB*w201. Only 3 animals (GJ48 in the C274/AT-2 SIV group, GJ57 in the polyICLC/AT-2 SIV group and DA47 in the control group) carried the B*03 and/or B*04 alleles, which have been associated with slow disease progression.
Having followed the animals for 4 months after cessation of ART and established that the polyICLC/AT-2 SIV vacinees showed better control of viral replication than the other groups, we evaluated the effects of subsequent immunization with the polyICLC/AT-2 SIV vaccine in the absence of ART in the animals initially vaccinated with polyICLC/AT-2 SIV, as well as the C274/AT-2 SIV vaccinated and control animals. Animals in each original treatment group were immunized with the polyICLC/AT-2 SIV vaccine, as described for the initial vaccinations. The two control animals (EL02 and GJ65) that had not responded to ART were excluded, having already been euthanized. All animals received polyICLC/AT-2 SIV at 20, 26, 32 and 38 weeks post-ART and followed up for 3 months after the final immunization.
Plasma viremia appeared unaffected by the polyICLC/AT-2 SIV immunizations in the absence of concomitant ART (
(A) Plasma viral loads were determined before, during and after treatment. Each symbol indicates an individual animal. Arrows indicate immunization time-point. (B) The average viremia (geometric means) for each group (±SEM) is shown. Asterisks denote the statistically significantly lower virus levels in the polyICLC group compared to the original C274 and/or control groups; p<0.04 for polyICLC vs C274 at weeks 23–46, p<0.03 for polyICLC vs control at weeks 20–34, for good ART responders. (C) Average AUC is shown for each group (±SEM) for the time period shown in panel A. Asterisks mark the statistically significant differences: polyICLC vs C274 at weeks 16–34, p<0.03; polyICLC vs control at weeks 16–34, p<0.03; polyICLC vs C274 at weeks 34–46, p<0.05. Each group's original treatment under ART is indicated in each panel.
More extensive examination of the adaptive immunity was carried out in the hope of identifying the responses involved in the improved long term control of virus replication in the original polyICLC/AT-2 SIV vacinees. In all animals the frequency of SIV-specific IFNγ producing cells measured in blood was unaltered by the polyICLC treatment in the absence of ART (
The average SIV-specific (to AT-2 SIV) IFNγ ELIPSOT responses during the vaccination are illustrated (±SEM). SIV specific responses were determined by subtracting the appropriate MV response.
We also examined the immune responses by ICS (
CD4+ | |||||||||||
TNFα | IL-2 | IFNγ | IL-17 | TNFα + IL-2 | TNFα + IFNγ | TNFα + IL-17 | IL-2 + IFNγ | IL-2 + IL-17 | IFNγ + IL-17 | ||
GJ39 | PBMC | + | - | + | - | - | + | - | - | - | - |
LN | + | - | - | + | + | + | + | + | - | + | |
GJ47 | PBMC | - | - | - | - | - | - | - | - | - | - |
LN | + | - | - | - | + | - | - | - | - | - | |
GJ48 | PBMC | + | - | + | + | - | + | + | - | + | + |
LN | |||||||||||
GJ50 | PBMC | + | -- | + | - | + | + | - | + | - | - |
LN | + | + | + | + | - | - | - | + | - | - | |
GJ53 | PBMC | + | + | + | + | + | + | + | - | + | + |
LN | + | + | - | - | + | - | - | - | - | - | |
BG93 | PBMC | ||||||||||
LN | - | - | + | - | - | - | - | - | - | - | |
GJ55 | PBMC | + | + | + | + | + | + | + | + | + | + |
LN | |||||||||||
GJ57 | PBMC | + | - | + | - | - | + | - | + | - | - |
LN | + | - | + | - | - | - | + | - | - | - | |
CC48 | PBMC | ||||||||||
LN | + | - | - | - | - | - | - | - | - | - | |
DV67 | PBMC | + | - | - | - | - | - | - | - | - | - |
LN | + | + | - | + | + | + | + | + | + | + | |
DD94 | PBMC | - | - | - | - | - | - | - | - | - | - |
LN | + | + | + | + | - | + | + | + | - | + | |
GJ97 | PBMC | - | - | - | - | - | - | - | - | - | - |
LN | + | + | + | + | + | + | + | + | + | + | |
DA47 | PBMC | + | + | + | + | + | + | + | + | + | + |
LN | + | + | - | + | - | - | + | - | - | - |
The expression of four cytokines was measured at the end of the study (week 46 post ART) by ICS of PBMC and lymph node (LN) cells. An average value of >0.01% was taken as a positive. Plus-signs indicate positivity, minus-signs indicate negativity, whereas white cells indicate that a value was not determined for that condition. Single and double cytokine combinations are shown. No triple or quadruple cytokine producing cells were detected. SIV-specific responses were measured by subtracting the corresponding MV response. CD4+ T cell cytokine production is shown.
CD4− | |||||||||||
TNFα | IL-2 | IFNγ | IL-17 | TNFα + IL-2 | TNFα + IFNγ | TNFα + IL-17 | IL-2 + IFNγ | IL-2 + IL-17 | IFNγ + IL-17 | ||
GJ39 | PBMC | + | - | - | - | - | - | - | - | - | - |
LN | |||||||||||
GJ47 | PBMC | + | + | - | - | - | + | - | - | - | - |
LN | |||||||||||
GJ48 | PBMC | ||||||||||
LN | |||||||||||
GJ50 | PBMC | + | - | - | - | - | - | - | - | - | - |
LN | - | - | - | + | - | - | + | - | + | - | |
GJ53 | PBMC | + | + | - | + | + | - | - | + | - | |
LN | + | + | + | + | + | + | + | + | + | + | |
BG93 | PBMC | - | - | + | - | - | - | - | - | - | - |
LN | + | + | + | + | - | + | + | - | + | + | |
GJ55 | PBMC | + | + | + | - | + | + | + | + | - | + |
LN | |||||||||||
GJ57 | PBMC | - | - | - | - | - | - | - | - | - | - |
LN | - | - | - | - | - | - | - | - | - | - | |
CC48 | PBMC | ||||||||||
LN | + | - | - | + | - | - | + | - | - | - | |
DV67 | PBMC | + | + | - | + | + | - | + | - | + | - |
LN | + | + | - | - | + | + | + | - | + | + | |
DD94 | PBMC | + | - | - | - | - | - | - | - | - | - |
LN | + | + | - | - | + | + | + | + | + | + | |
GJ97 | PBMC | - | - | - | - | - | + | + | + | - | + |
LN | - | - | - | - | + | + | - | + | - | + | |
DA47 | PBMC | + | + | + | + | + | + | + | + | + | + |
LN | + | - | - | - | - | - | - | - | - | - |
The expression of four cytokines was measured at the end of the study (week 46 post ART) by ICS of PBMC and lymph node (LN) cells. An average value of >0.01% was taken as a positive. Plus-signs indicate positivity, minus-signs indicate negativity, whereas white cells indicate that a value was not determined for that condition. Single and double cytokine combinations are shown. No triple or quadruple cytokine producing cells were detected. SIV-specific responses were measured by subtracting the corresponding MV response. CD4− T cell cytokine production is shown.
This study aimed to investigate the combination of AT-2 inactivated SIV with 2 TLR-binding adjuvants, C274 and polyICLC, as a mucosal therapeutic vaccine for SIV-infected macaques. We have previously shown AT-2 SIV to be partially effective in preventing SIV infection in macaques
ART consisted of a two-drug regimen that has been previously shown to be partially effective in reducing plasma viremia
After the test group animals were vaccinated four times during ART, therapy was withdrawn. As expected from previous studies in macaques
Since the polyICLC/AT-2 SIV vaccination showed promise when administered under ART we were interested to assess how it performed in the absence of ART. Unfortunately, polyICLC/AT-2 SIV vaccination did not reduce plasma virus levels in any of the groups in the absence of ART. However, there were no adverse affects of the vaccination either. SIV-infected Rhesus macaques develop symptoms of simian AIDS relatively rapidly
We attempted to determine the correlates of immune control of viral replication after the effective vaccination under ART as well as after subsequent vaccination without ART, measuring a variety of adaptive and innate responses in blood and tissues, but most responses were comparable between groups at the time-points measured. However, we were able to detect significantly higher neutralizing Ab activity in polyICLC/AT-2 SIV vaccinees, after the end of ART, which may account for some of the protective effect of the vaccine. It is well established that high neutralizing Ab activity is inversely correlated with plasma viremia
The role of Tregs in HIV infection is uncertain. They have been shown to suppress HIV-specific T cell responses
Another level of immune control might be dictated by the MHC allele of individual monkeys. Certain macaque MHC alleles, like Mamu-A*01
Previous studies demonstrated beneficial therapeutic vaccination for SIV infection, administered intramuscularly
No correlation of T cell parameters with virus control (A) T cell subsets and activation markers (in the CD3+CD4+ and CD3+CD4− gates) were measured in PBMC, lymph nodes (LN), jejunum and ileum of all available animals at the time of necropsy (n = 6 in C274/AT-2 SIV group, n = 6 in polyICLC/AT-2 SIV group). Tregs were defined as CD25+FoxP3+; naïve T cells as CD95−; central memory T cells (TCM) as CD95+CD28+ and effector memory T cells (TEM) as CD95+CD28−. Average percentages for each group are shown (±SEM). (B) PD-1 surface expression (CD4+PD1+ and CD4−PD1+) was measured in PBMC and LN of all animals at the end of the study, by flow cytometry. Average percentages of PD-1 expressing cells per vaccination group are shown (±SEM).
(1.45 MB TIF)
Similar cytokine, chemokine and Treg marker expression in tissues, between groups. RNA was extracted from tissue cell pellets (10×106 cells/pellet). RT-PCR was performed for a range of cytokines and chemokines, as well as Treg markers. (A) Jejunum and LN cell RNA expression of cytokines and Treg markers. (B) Jejunum cell RNA expression of cytokines and chemokines. mRNA levels are shown in arbitrary units (AU) which were calculated by subtracting the threshold cycle (CT) number of the gene of interest from 100, then normalizing by dividing with (100-CT) of housekeeping gene β-actin, then multiplying by 100. Means (±SEM) of 6 C274/AT-2 SIV and 4 polyICLC/AT-2 SIV animals are shown for LN and 6 C274/AT-2 SIV and 3 polyICLC/AT-2 SIV animals for jejunum.
(2.46 MB TIF)
174xCEM cells were obtained from the NIH AIDS Research and Reference Reagent Program, courtesy of Peter Cresswell. We thank Julian Bess, William Bohn, Jeremy Miller, Terra Schaden-Ireland, Rodman Smith, Robert Imming and Elena Chertova, at NCI-Frederick, for producing, inactivating, purifying and characterizing AT-2 SIV and MV preparations. We thank Jason Marshall and Gary Van Nest from Dynavax Technologies for the ODNs and Norbert Bischofberger from Gilead Sciences for providing the antiretroviral drugs. We would like to acknowledge the Rockefeller University Flow Cytometry Resource Center for flow cytometry assistance and the veterinary staff at the TNPRC for their continued support. We thank members of our laboratory for their assistance in editing the manuscript and continued help during the course of this study and particularly Nina Derby and Ariel Martinez for their assistance with PCR. Additional thanks go to Daniel Gawarecki for assistance with statistics.