Control of Viremia and Prevention of AIDS following
Immunotherapy of SIV-Infected Macaques with PeptidePulsed Blood
Robert De Rose1, Caroline S. Fernandez1, Miranda Z. Smith1, C. Jane Batten1, Sheilajen Alcântara1,
Vivienne Peut1, Erik Rollman1, Liyen Loh1, Rosemarie D. Mason1, Kim Wilson2, Matthew G. Law3,
Amanda J. Handley1,4¤, Stephen J. Kent1*
1 Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia, 2 National Serology Reference Laboratory, Fitzroy, Victoria,
Australia, 3 National Centre for HIV Epidemiology and Clinical Research, University of New South Wales, Sydney, New South Wales, Australia, 4 Opal Therapeutics Pty Ltd,
Melbourne, Victoria, Australia

Abstract
Effective immunotherapies for HIV are needed. Drug therapies are life-long with significant toxicities. Dendritic-cell based
immunotherapy approaches are promising but impractical for widespread use. A simple immunotherapy, reinfusing fresh
autologous blood cells exposed to overlapping SIV peptides for 1 hour ex vivo, was assessed for the control of SIVmac251
replication in 36 pigtail macaques. An initial set of four immunizations was administered under antiretroviral cover and abooster set of three immunizations administered 6 months later. Vaccinated animals were randomized to receive Gag
peptides alone or peptides spanning all nine SIV proteins. High-level, SIV-specific CD4 and CD8 T-cell immunity was induced
following immunization, both during antiretroviral cover and without. Virus levels were durably ,10-fold lower for 1 year inimmunized animals compared to controls, and a significant delay in AIDS-related mortality resulted. Broader immunity
resulted following immunizations with peptides spanning all nine SIV proteins, but the responses to Gag were weaker incomparison to animals only immunized with Gag. No difference in viral outcome occurred in animals immunized with all SIV
proteins compared to animals immunized against Gag alone. Peptide-pulsed blood cells are an immunogenic and effective
immunotherapy in SIV-infected macaques. Our results suggest Gag alone is an effective antigen for T-cell immunotherapy.
Fresh blood cells pulsed with overlapping Gag peptides is proceeding into trials in HIV-infected humans.
Citation: De Rose R, Fernandez CS, Smith MZ, Batten CJ, Alcântara S, et al. (2008) Control of Viremia and Prevention of AIDS following Immunotherapy of SIVInfected Macaques with Peptide-Pulsed Blood. PLoS Pathog 4(5): e1000055. doi:10.1371/journal.ppat.1000055
Editor: Richard A. Koup, National Institutes of Health-NIAID, United States of America
Received January 24, 2008; Accepted April 1, 2008; Published May 2, 2008
Copyright: ß 2008 De Rose et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Supported by OPAL Therapeutics Pty Ltd, the Australian Centre for HIV and Hepatitis Research, and Australian National Health and Medical ResearchCouncil grants 299907, 251653, 454553, 359281. The funders played no role in the design and conduct of the study, nor in the collection, analysis andinterpretation of the data, nor in the preparation, review or approval of the manuscript.
Competing Interests: SJK spun out a venture capital–backed biotechnology start-up company, OPAL Therapeutics Pty Ltd, to pursue this technology towards
clinical trials. SJK, CJB and the University of Melbourne hold shares in this company. AJH has been an employee of OPAL Therapeutics.
* E-mail: skent@unimelb.edu.au¤ Current address: Medicines Development Ltd, Melbourne, Victoria, Australia

and CD8 T-cell responses to both structural and regulatory
proteins can be induced, and peptide antigens are simple tomanufacture to high purity. This study assessed whether OPAL
vaccination improves the outcome of SIV-infected monkeys.
Considerable debate exists regarding the most effective antigens
to target for T-cell based therapeutic HIV vaccination. It has been
widely believed that broader immunity to multiple proteins would
be more efficacious [10,11]. In contrast, recent studies highlightthe effectiveness of Gag-specific T cell immunity in comparison to
T cell immunity to other antigens. We therefore also assessed
whether narrower responses induced only to SIV Gag are aseffective as more broadly targeting all 9 SIV proteins.

Introduction
Several attempts at immunotherapy of HIV using moreconventional vaccines have thus far been poorly immunogenic
and weakly efficacious in human trials [1,2,3,4]. The use of
professional antigen-presenting cells such as dendritic cells todeliver HIV immunotherapies has shown strong immunogenicity
efficacy in macaques and pilot humans studies but is limited tohighly specialized facilities [5,6,7]. A simple intermittent immunotherapy that reduces the need for long-term antiretroviraltherapy (ART) would be a quantum advance in treating HIV.
We recently reported the robust T-cell immunogenicity oftreating unfractionated whole blood or peripheral blood mononuclear cells (PBMC) with overlapping peptides of SIV, HIV-1 or
hepatitis C virus in outbred pigtail monkeys [8,9]. We termed thissimple immunotherapy OPAL (Overlapping Peptide-pulsed
Autologous ceLls). This technique is attractive since there is no
prolonged ex vivo culture of antigen-presenting cells, robust CD4

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Materials and Methods
Animals
Juvenile pigtail macaques (Macaca nemestrina) free from Simian
retrovirus type D were studied in protocols approved by1

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OPAL Immunotherapy for SIV

peptides would result in some level of immune activation and drive
higher viral loads in controls, artificially magnifying any reductionsin the active treatment groups, (c) reinfusion of control peptide
pulsed cells might have obscured any unexpected safety problemsof the procedure.

Author Summary
Effective immunotherapies for HIV are needed. We
assessed a simple technique, reinfusion of fresh blood
cells incubating with overlapping SIV peptides (Overlapping Peptide-pulsed Autologous ceLls, OPAL), in 36
randomly allocated SIV-infected monkeys. We analyzed
this therapy for the stimulation of immunity, control ofvirus levels, and prevention of AIDS. The OPAL immunotherapy was safe and stimulated remarkable levels of T-cell
immunity. Levels of virus in vaccinated monkeys were 10fold lower than in controls, and this was durable for over
1 year after the initial vaccinations. The immunotherapy
resulted in fewer deaths from AIDS. We conclude this is apromising immunotherapy technique. Trials in HIV-infected humans of OPAL therapy are planned.

Immunology assays
SIV-specific CD4 and CD8 T-cell immune responses wereanalysed by expression of intracellular IFNc as previously
described [21]. Briefly, 200 ml whole blood was incubated at
37uC with 1 mg/ml/peptide overlapping 15mer SIV peptide pools(described above) or DMSO alone and the co-stimulatory
antibodies anti-CD28 and anti-CD49d (BD Biosciences/Pharmingen San Diego CA) and Brefeldin A (10 mg/ml, Sigma) for 6 h.
Anti-CD3-PE, anti-CD4-FITC and anti-CD8-PerCP (BD, clones
SP34, M-T477 and SK1 respectively) antibodies were added for
30 min. Red blood cells were lysed (FACS lysing solution, BD) andthe remaining leukocytes permeabilized (FACS Permeabilizing
Solution 2, BD) and incubated with anti-human IFNc-APC
antibody (BD, clone B27) prior to fixation and acquisition (LSRII,
BD). Acquisition data were analyzed using Flowjo version 6.3.2(Tree Star, Ashland, OR). The percentage of antigen-specificgated lymphocytes expressing IFNc was assessed in both
CD3+CD4+ and CD3+CD8+ lymphocyte subsets. Responses tothe immunodominant SIV Gag CD8 T-cell epitope KP9 in ManeA*10+ animals were assessed by a Mane-A*10/KP9 tetramer as
described [13]. Total peripheral CD4 T-cells were measured as aproportion of lymphocytes by flow cytometry on fresh blood.

institutional animal ethics committees and cared for in accordancewith Australian National Health and Medical Research Council
guidelines. All pigtail macaques were typed for MHC class I alleles
by reference strand mediated conformational analysis and thepresence of Mane-A*10 confirmed by sequence specific primer
PCR as described [12,13]. 36 macaques were injected intravenously with 40 tissue culture infectious doses of SIVmac251 (kindly
provided by R. Pal, Advanced Biosciences, Kensington, MD) as
described previously [14,15] and randomized into 3 groups of 12
animals (OPAL-Gag, OPAL-All, Controls) 3 weeks later. Randomization was stratified for peak SIV viral load at week 2, weight,
gender and the MHC I gene Mane-A*10 (which is known toenhance immune control of SIV) [15]. Animals received
subcutaneous injections of dual anti-retroviral therapy withtenofovir and emtricitibine (kindly donated by Gilead, Foster
City, CA; both 30 mg/kg/animal) for 7 weeks from week 3: daily
from weeks 3–5 post-infection and three times per week fromweeks 6–10. This dual ART controls viremia in the majority of
SIV-infected macaques [16,17,18,19,20].

Virology assays
Plasma SIV RNA was quantitated by real time PCR on 140 mlof plasma at the University of Melbourne (lower limit ofquantitation 3.1 log10 copies/ml) at all time-points using a
TaqMan probe as previously described [21,22] and, to validate
these results with a more sensitive assay, on pelleted virions from
1.0 mL of plasma at the National Cancer Institute (lower limit ofquantitation 1.5 log10 copies/ml) as previously described [23].

Immunizations
Two animal groups (OPAL-Gag and OPAL-All) were immunized with OPAL immunotherapy using PBMC as previously
described [8]. Briefly, peripheral blood mononuclear cells (PBMC)
were isolated over Ficoll-paque from 18 ml of blood (anticoagulated with Na+-Heparain). All isolated PBMC (on average 24
million cells) were suspended in 0.5 ml of normal saline to which
either a pool of 125 SIVmac239 Gag peptides or 823 peptides
spanning all SIVmac239 proteins (Gag, Pol, Env, Nef, Vif, Tat, Rev,
Vpr, Vpx) were added at 10 mg/ml of each peptide within thepool. Peptides were 15mers overlapping by 11 amino acids at.80% purity kindly provided by the NIH AIDS reagent
repository program (catalog numbers 6204, 6443, 6883, 644850, 6407, 8762, 6205). To pool the peptides, each 1 mg vial oflyophilised 15mer peptide was solubilized in 10–50 ml of pure
DMSO and added together. The concentration of the SIV Gag
and All peptide pools was 629 and 72 mg/ml/peptide respectively,
although each peptide was pulsed onto cells at 10 mg/ml
regardless of vaccine type. The peptide-pulsed PBMC were heldfor 1 hr in a 37uC waterbath, gently vortexed every 15 minutes
and then, without washing, reinfused IV into the autologous
animal. Peptide concentrations and timing of incubation wereadapted from effective stimulation of T cell responses in vitro.
Control macaques did not receive vaccine treatment. This wasdone since (a) we had not previously observed any significant VLchanges with non HIV/SIV peptide sets ([8,9] and unpublisheddata), (b) reinfusion of blood cells pulsed with irrelevant sets of
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Endpoints/statistical analyses
The primary endpoint was the reduction in plasma SIV RNA in
OPAL-immunized animals compared to controls by timeweighted area-under-the-curve (TWAUC) for 10 weeks following
withdrawal of ART (i.e. samples from weeks 12 to 20). Thissummary statistical approach is recommended for studies such asthese involving serial measurements [24]. We compared bothactive treatment groups (OPAL-Gag and OPAL-All) to controls
separately and together. The primary analysis was restricted toanimals that controlled viremia on the ART at week 10 (VL,3.1
log10 copies/ml), since control of VL is an important predictor of
the ability of animals to respond to immunotherapies [8,25]. A
pre-planned secondary virologic endpoint was studying all live
animals adjusting for both VL at the end of ART (week 10) andMane-A*10 status. Group comparisons used two-sample t-tests forcontinuous data, and Fisher’s exact test for binary data. Survival
analyses utilised Cox-regression analyses.

Power calculation
Prior to initiating the study, we estimated the standard deviationof the return of VL after treatment interruption would beapproximately 0.8 log10 copies of SIV RNA/mL plasma[5,16,17,18,19,20]. In this intensive study we estimated that 2 of
the 12 monkeys within a group may have confounding problems
such as incomplete response to ART or death from acute SIV
2

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OPAL Immunotherapy for SIVThis study was conducted according to a pre-written protocol
using Good Laboratory Practice Standards from the Australian
Therapeutic Goods Administration as a guide. Protocol deviations
were minor and did not affect the results of the study. Partial data
audits during the study did not raise any concerns about the study
conduct.

responses were reduced in comparison to only immunizing with
Gag peptides.
Although the short linear peptides were primarily used toinduce T cell immunity, we also studied serial plasma samples for
SIV-specific antibodies. All animals seroconverted following SIV
infection, as shown by Western Blot (Fig. 3A). No significant
enhancement of Gag or Env antibody responses occurred with the
OPAL vaccinations (Fig. 3B, C). There was a dip in mean Gag
antibody responses during the period of ART in all groups
consistent with reduced viral antigen during this period. In
addition to the lack of difference in mean Gag (p26) or Env (gp36)
responses shown in Figure 3B and 3C, there were also no
significant different antibody responses to p16, p68, gp125 and
gp140 across the vaccine groups (not shown).

Results

Virologic outcome following initial vaccinations

infection. A 10 control vs 10 active treatment comparison yields
80% power (p = 0.05) to detect a 1.0 log10 difference in TWAUC
VL over the first 10 weeks. An estimated comparison of 10 control
vs all 20 actively treated animals (OPAL-Gag plus OPAL-All) gave
80% power to detect differences of 0.87 log10 copies/ml VL
reduction.

Study conduct

The 7-week period of ART controlled VL to below 3.1 log10
copies/ml in 26 of the remaining 32 animals by week 10 (Table
S1). The pre-defined (per-protocol) primary VL endpoint analyses
was performed on animals controlling viremia on ART (26animals). The 6 animals that failed to control viremia on ART had
higher peak VLs at week 2 (mean6SD of 7.7460.33 compared to
6.9460.52 for animals controlling viremia on ART, p,0.001) andhigher VL following ART withdrawal (5.9860.53 vs 4.2860.90,
p,0.001). Control of VL is likely to be important in achieving
optimal results from immunotherapy of infected macaques [8,20].
The primary endpoint comparison of VL between combined
OPAL-All and OPAL-Gag treatment groups in the 10 weeks after
ART withdrawal was 0.5 log10 copies/ml lower than controls(p = 0.084, Fig 4, Table 1). Each vaccination group (OPAL-All and
OPAL-Gag) had very similar reductions in VL. By 6 months after
ART withdrawal, the mean difference in VL between control and
OPAL-immunized groups was 0.93 log10 copies/ml (p = 0.028,
Table 1).
As a secondary endpoint, we also analysed all 32 remaining
animals by adjusting for VL control on ART and Mane-A*10
status. There was a significant difference in VL between controls
and vaccinated macaques with these analyses at both 10 and
26 weeks off ART (p = 0.050, 0.016 respectively, Table 1).
To confirm the virologic findings using a sensitive independent
VL assay, frozen plasma (1 ml) from study week 32 was shipped to
the National Cancer Institute (NCI) in Maryland, USA. Drs M
Piatak and J Lifson kindly analysed the samples for SIV RNA
blindly using an assay with a limit of quantitation of 1.5 log10
copies/ml (Table S1) [23]. The University of Melbourne and NCI
assays were tightly correlated (r = 0.97, p,0.001) and showed analmost identical mean reduction in viremia in vaccinees compared
to controls at this time (0.82 vs 0.88 log10 copies/ml respectively).

OPAL Vaccinations
OPAL immunotherapy was studied in SIV-infected pigtail
macaques receiving ART. Pigtail macaques have at least an
equivalently pathogenic course of SIV infection as alternate rhesus
macaque models [14,26]. Thirty-six macaques were infected with
SIVmac251 and 3 weeks later treatment with the antiretrovirals
tenofovir and emtricitabine for 7 weeks was initiated. The animals
were randomly allocated to 3 groups stratified by peak plasma SIV
viral load (VL), Mane-A*10 status (an MHC class I gene that
improves VL in SIV-infected pigtail macaques [15]), weight andgender. Macaques were immunized 4 times under the cover ofantiretroviral therapy (weeks 4, 6, 8, 10) with autologous fresh
PBMC mixed for 1 hour ex vivo with 10 mg/ml/peptide of either
125 overlapping SIV Gag 15mer peptides only (OPAL-Gag), 823
SIV 15mer peptides spanning all 9 SIV proteins (OPAL-All) or unimmunized. The macaques were initially followed for 26 weeks
after ceasing ART on week 10.
All 36 macaques became infected following SIVmac251 exposureand had a mean peak VL of 7.1 log10 copies/ml (Table S1). Prior
to vaccination, 4 animals died during acute SIV infection withdiarrhoea, dehydration, lethargy, anorexia and weight loss. The
vaccinations were well tolerated, with no differences in mean
weights, haematology parameters, or clinical observations in
OPAL immunized animals compared to controls (data not shown).

Immunogenicity
There was striking SIV-specific CD4+ and CD8+ T-cell
immunogenicity after the course of vaccination in the OPAL
immunized animals. Mean Gag-specific CD4 and CD8 T-cell
responses 2 weeks after the final immunization were 3.0% and1.9% of all CD4 and CD8 T cells respectively in the OPAL-Gag
group. Mean Gag-specific CD4 and CD8 T-cell responses 2 weeks
after the final immunization were 0.84% and 0.37% in the OPALAll group and 0.15% and 0.29% in controls (Fig. 1A, B). The Gagspecific T cells in the OPAL-All immunized animals, but notcontrol or OPAL-Gag only immunized animals, also had elevatedT-cell responses to all other SIV proteins. Mean Env, Pol andcombined regulatory protein-specific CD4/CD8 responses were2.5%/11.8%, 0.8%/0.3% and 1.5%/2.4% respectively in the
OPAL-All group compared to #0.4% for all CD4/8 responses to
non-Gag antigens in control and OPAL-Gag groups (Fig. 1C, D
and Fig. 2). The kinetics of induction of non-Gag CD4 and CD8 T
cell responses in the OPAL-All group was similar for induction of
Gag-specific T cell immunity. Stronger CD8 T-cell responses to
non-Gag proteins correlated with reduced CD8 T-cell responses toGag (Fig. 1E). Thus, although a larger number of SIV proteins
were recognized in the OPAL-All immunized animals, Gag
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Durability of OPAL immunotherapy
To further assess the durability of SIV control and prevention of
disease with OPAL immunotherapy, we re-boosted all 32 animals
in the same randomized groups 3 times with the identicalprocedure (at week 36, 39, 42) without ART cover and followed
the animals for an additional 6 months. Despite the lack of ART
cover, SIV-specific T cell immunity was dramatically enhanced in
immunized animals 2 weeks after the last vaccination, similarly tothe primary vaccination (Figs 1, 2). The T cell responses to Gag
were again highest in the OPAL-Gag group with broader
responses in the OPAL All group. The pattern of enhancementof T cell immunity was similar for the first and second vaccination
sets (Figs 1, 2).
We again sampled plasma for viral load every 3–6 weeks. To
account for the death of animals from AIDS, we used a ‘‘last3

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OPAL Immunotherapy for SIV

Figure 1. T-cell immunogenicity of OPAL vaccination. SIV Gag-specific CD4 (A) and CD8 (B) T-cells expressing IFNc were studied over time byintracellular cytokine staining. Mean6standard error of vaccine groups compared to control unvaccinated animals (circles) is shown. The primary OPAL
vaccinations of macaques (arrows, weeks 4, 6, 8 and 10 after SIVmac251 infection) consisted of autologous PBMC pulsed with either overlapping SIV Gag
15mer peptides (OPAL-Gag, triangles) or peptides spanning all 9 SIV proteins (OPAL-All, squares). Initial vaccinations were given under the cover ofantiretroviral treatment (ART). Animals were re-boosted with OPAL immunotherapy in the same randomised groups, without ART, at weeks 39, 42 and 42.
At week 12, two weeks after the last vaccination, CD4 (C) and CD8 (D) T-cell responses to pools of overlapping peptides spanning SIV Gag, Env, Pol or
combined Regulatory/Accessory proteins (Nef, Tat, Rev, Vif, Vpx, Vpr [Reg]) were assessed in all animals by intracellular cytokine staining. In addition,
responses to a SIV Gag CD8 T-cell epitope KP9, were assessed by a Mane-A*10/KP9 tetramer. Mean6standard error of vaccine groups is shown along with 2sided t-test p values of ,0.10. (E) SIV Gag specific CD8 T-cell responses correlated inversely with CD8 T-cell responses to the summation of non-Gag(Env+Pol+Regulatory protein) responses across all 21 live OPAL-immunized animals. The animals with .50% CD8 T-cell responses to the combined pool
had total responses of 50.4% and 54.5%, primarily to Env (50.1% and 54.2% respectively). Spearman rank correlation is shown.
doi:10.1371/journal.ppat.1000055.g001

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OPAL Immunotherapy for SIV

Figure 2. Non-Gag T cell immunogenicity of OPAL Vaccination. SIV-specific CD4 and CD8 T-cells expressing IFNc were studied over time byintracellular cytokine staining to Env (A, B), Pol (C, D) and a pool of overlapping peptides spanning combined Regulatory/Accessory proteins(RTNVVV, E, F). Mean6standard error of vaccine groups compared to control unvaccinated animals (circles) is shown. Four initial vaccinations were
given weeks 4–10 and a second set of 3 immunizations given weeks 36–42 as shown in Fig 1A.
doi:10.1371/journal.ppat.1000055.g002

observation carried forward’’ analysis for missing VL data.
Significant viral control was maintained throughout the follow
up period of just over 1 year off ART (Fig 4A, Table 1). In animals
which controlled VL on ART, there was a mean 0.98 log10

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copies/ml difference between controls and vaccinees 54 weeks
after coming off ART (p = 0.019 for time-weighted analysis).
Twelve of the remaining 32 animals developed incipient AIDS
and were euthanised during the extended follow up. All 6 animals

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OPAL Immunotherapy for SIV

Figure 3. SIV-specific antibody responses. All 32 live macaques had serial measurements of SIV-specific antibodies utilizing HIV-2 Western Blot
strips. (A) Representative examples of the evolution of the Western Blot profiles of a macaque within each vaccine group. A positive control sample
from HIV-2 infected (+) and uninfected (2) human subjects are shown at the right of the panel. (B) Mean6standard error densitometry
measurements of Gag (anti-p26) responses and (C) Env (anti-gp36) responses over time.
doi:10.1371/journal.ppat.1000055.g003

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Figure 4. Efficacy of OPAL immunotherapy. Antiretroviral therapy (ART) was withdrawn at week 10, after the last vaccination, and (A) plasma
SIV RNA followed. The 26 animals that controlled viremia on ART are illustrated with mean6standard error of vaccine groups. (B) Survival of these 26
vaccinated and controls animals is shown. P values represent the difference between controls and the combined vaccine groups (see Table 1).
doi:10.1371/journal.ppat.1000055.g004

that did not control viremia on ART required euthanasia. Of the 6euthanised animals which did control viremia on ART, 5 were in
the control group and one in the OPAL-Gag group. OPAL
immunotherapy resulted in a survival benefit, analysing either the
26 animals that controlled viremia on ART (p = 0.053, Fig 4B,
Table 1) or all 32 animals, adjusted for Mane-A*10 status andcontrol of viremia on ART (p = 0.02, Table 1).

findings on OPAL immunotherapy were observed despite thevirulent SIVmac251-pigtail model studied [14] and provide strong
proof-of-principle for the promise of this immunotherapy
technique.
The OPAL immunotherapy approach is simpler than manyother cellular immunotherapies, particularly the use of dendritic
cells. The use of DNA, CTLA-4 blockade and viral vector based
approaches are also now showing some promise in macaquestudies [17,27], although such approaches have not yet beentranslated into human studies. This study added peptides to
PBMC, however we have shown an even simpler technique,adding peptides to whole blood is also highly immunogenic, atechnique that will be more widely applicable ([8] and unpublishedstudies).
This is one of the largest therapeutic SIV vaccine studies yetreported. Although it may have been ideal to have studied

Discussion
In summary, OPAL immunotherapy, either using overlapping Gag SIV peptides or peptides spanning the whole SIV
proteome was highly immunogenic and resulted in significantly
lower viral loads and a survival benefit compared to unvaccinated controls. The virologic efficacy in OPAL-immunizedmacaques was durable for 12 months after ART cessation. Our
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6 of the 32 animals failed to control viremia on ART.
VL values reductions are log10 copies/ml compared to controls. Values shown reflect time-weighted area-under-the-curve VL between vaccinated animals and controls after coming off ART, and absolute mean reduction at theend of the period in parentheses.
c
12 animals died after week 41; the mean of the 2 last VL observations were carried forward to estimate differences in VL to week 64.
dSurvival p-value reflect Cox-regression analysis.
eNone of the 8 OPAL-All vaccinated animals that had VL undetectable on ART died compared to 5 of 10 controls – this comparison did not permit an estimate of significance of this comparison.
doi:10.1371/journal.ppat.1000055.t001

irrelevant peptide-pulsed autologous cells as an additional control
group, we were concerned that this may have magnified thetherapeutic effect or obscured any safety concerns. In the end, thevaccination process was both safe and effective.
How well the findings on OPAL immunotherapy translate tohumans with acute HIV-1 infection will be determined by clinical
trials. Virus-specific CD4 T cells are typically very weak in HIVinfected humans or SIV-infected macaques; dramatic enhancement of these cells were induced by OPAL immunotherapy andthis may underlie its efficacy [28]. We measured IFNc-producing
T cells in this study since we had not developed polyfunctional ICS
assays prior to initiating the study. However, recent cross-sectional
polyfunctional ICS assays suggests OPAL immunotherapy canalso induce T cells capable of also expressing the cytokines TNFa
and IL-2, the chemokine MIP1b and the degranulation marker
CD107a (unpublished data).
A ,1.0 log10 reduction in VL would result in a substantial
delay in progressive HIV disease in humans and allow areasonable time period without the requirement to reintroduce
ART [29] if these findings are confirmed in human trials. Boththe control and vaccinated macaques were treated with ART
early in this study (3 weeks after infection), which alone can beassociated with a transiently improved outcome in humans [30].
None-the-less, a massive loss of CD4+ T cells in the gut occurs
within 2 weeks of infection [31]. Although it may be challenging
to identify humans within 3 weeks of infection, this is when HIV1 subjects typically present with acute infection. The durable
control of viremia exhibited by the vaccinated animals isinteresting and consistent with other recent macaque studies[27], suggesting the need for re-immunization may not be
substantial. We cannot attribute the durable control of viremia to
the second set of immunizations; there was only a marginal, nonsignificant, increase in the difference in VL between OPAL
vaccinees and controls before and after the second immunization
series. Further studies are required to address the timing andbenefit of ART cover during boosting immunizations with OPAL
immunotherapy.
Control of viremia was similar for the OPAL-Gag and OPALAll groups. Gag-specific CD4 and CD8 T-cell responses in OPALGag animals 5.1- and 3.5-fold greater than those in the OPAL-All
animals, despite an identical dose of Gag overlapping peptides.
This suggests antigenic competition between peptides from Gag
and the other SIV proteins. Inducing immunodominant non-Gag
T-cell responses by multi-protein HIV vaccines may limit thedevelopment of Gag-specific T-cell responses [21]. A large humancohort study demonstrated Gag-specific T-cell responses were themost effective in controlling HIV viremia [32]. Useful subdominant T cell responses may be particularly susceptible to dominant
non-Gag T cell responses [33,34]. The utility, if any, of inducing
T-cell responses to non-Gag proteins (i.e. excluding Gag peptides
from the vaccine antigens) can be addressed in future studies ofthis flexible vaccine technology. Therapeutic HIV vaccines may
not need to aim for maximally broad multi-protein HIV-specificimmunity.
OPAL immunotherapy with Gag peptides is proceeding into
initial trials in HIV-infected humans. Additional peptides canreadily be added into standard consensus strains mixes to covercommon strain or subtype variations between strains with thistechnology [35]. Additional technologies such as toggling variableamino acids peptides may provide further T cell immunogenicity
with this general technology [36]. Immunotherapy with peptides
delivered onto fresh blood may have potential applicability forother chronic viral diseases such as hepatitis C virus infection andsome cancers such as melanoma [37].

b

a

0.022

0.054
0.023

0.035

0.71 (0.60)

0.77 (0.72)

0.040

0.032

0.60 (0.64)

0.63 (0.69)
0.116

0.072
0.51 (0.53)

0.44 (0.54)
10 vs 11

11 vs 11
Individual vaccine arms OPAL-All vs controls

OPAL-Gag vs controls

0.212

0.020
0.011

0.069
0.81 (1.07)

0.74 (0.66)

0.080

0.61 (0.66)
0.050

0.016

0.67 (0.95)
0.262
0.57 (0.71)

0.47 (0.54)
21 vs 11

8 vs 10
OPAL-Gag vs controls

OPAL-Gag+All vscontrols
All animals, adjusted for Combined vaccine
Mane-A*10 status and VL groupat week 10 (n = 32)

NAe
0.066
0.79 (0.88)
Individual vaccine arms OPAL-All vs controls

8 vs 100.42 (0.74)

0.136

0.61 (0.90)

0.114

0.053
0.019
0.80 (0.98)
0.028
0.64 (0.93)
0.084
0.50 (0.73)
16 vs 10
Combined vaccinegroup
VL undetectable atweek 10a (n = 26)

OPAL- Gag+All vscontrols
Comparison
Animals analyzed

Table 1. Statistical analyses of VL and Survival.

nReduction in VL
2 sided t-test Reduction in VL
10 weeks off ARTb (p-value)
6 months off ART

2 sided t-test Reduction in VL(p-value)
1 year off ARTc

2 sided t-test(p-value)

Survival 1 year
off ART (p-value)d

OPAL Immunotherapy for SIV

8

May 2008 | Volume 4 | Issue 5 | e1000055

OPAL Immunotherapy for SIV

M. Miller for the antiretrovirals, and M. Piatak and J. Lifson for viral load
assays.

Supporting Information
Table S1 Viral load data
Found at: doi:10.1371/journal.ppat.1000055.s001 (0.08 MB PDF)

Author Contributions
Conceived and designed the experiments: SK. Performed the experiments:
RD CF MS CB SA VP ER LL RM KW AH SK. Analyzed the data: ML
SK. Wrote the paper: SK. Performed the statistical analyses: ML. Audited
the study: AH.

Acknowledgments
We thank J. Reece, B. Pratt, J. Stephany, J. Stewart, T. Amarasena, A.
Townsend, A. Chung, A. Brooks, J. Lin, K. Frost, K. Szalnowski, and R.
Goli at U Melbourne for expert assistance, R. Pal for the SIVmac251 stock,

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