Control of viremia and prevention of AIDS following immunotherapy of SIV-infected macaques with peptide-pulsed blood.
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 SIV(mac251) replication in 36 pigtail macaques. An initial set of four immunizations was administered under antiretroviral cover and a booster 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 approximately 10-fold lower for 1 year in immunized 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 in comparison 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.
- SourceAvailable from: Janka Petravic[Show abstract] [Hide abstract]
ABSTRACT: Resting CD4+ T cells are a reservoir of latent HIV-1. Understanding the turnover of HIV DNA in these cells has implications for the development of eradication strategies. Most studies of viral latency focus on viral persistence under antiretroviral therapy (ART). We studied the turnover of SIV DNA resting CD4+ T cells during active infection in a cohort of 20 SIV-infected pigtail macaques. We compared SIV sequences at two Mane-A1*084:01-restricted CTL epitopes using serial plasma RNA and resting CD4+ T cell DNA samples by pyrosequencing, and used a mathematical modeling approach to estimate SIV DNA turnover. We found SIV DNA turnover in resting CD4+ T cells was slow in animals with low chronic viral loads, consistent with the long persistence of latency seen under ART. However, in animals with high levels of chronic viral replication, turnover was high. SIV DNA half-life within resting CD4 cells correleated with viral load (p = 0.0052) at the Gag KP9 CTL epitope. At a second CTL epitope in Tat (KVA10) there was a trend towards an association of SIV DNA half-life in resting CD4 cells and viral load (p = 0.0971). Further, we found that the turnover of resting CD4+ T cell SIV DNA was higher for escape during early infection than for escape later in infection (p = 0.0084). Our results suggest viral DNA within resting CD4 T cells is more labile and may be more susceptible to reactivation/eradication treatments when there are higher levels of virus replication and during early/acute infection.PLoS ONE 04/2014; 9(4):e93330. · 3.53 Impact Factor
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ABSTRACT: HIV Gag-specific CD4+ and CD8+ T-cell responses are important for HIV immune control. Pulsing overlapping Gag peptides on autologous lymphocytes (OPAL) has proven immunogenic and effective in reducing viral loads in multiple pigtail macaque studies, warranting clinical evaluation. We performed a phase I, single centre, placebo-controlled, double-blinded and dose-escalating study to evaluate the safety and preliminary immunogenicity of a novel therapeutic vaccine approach 'OPAL-HIV-Gag(c)'. This vaccine is comprised of 120 15mer peptides, overlapping by 11 amino acids, spanning the HIV Gag C clade sequence proteome, pulsed on white blood cells enriched from whole blood using a closed system, followed by intravenous reinfusion. Patients with undetectable HIV viral loads (<50 copies/ml plasma) on HAART received four administrations at week 0, 4, 8 and 12, and were followed up for 12 weeks post-treatment. Twenty-three people were enrolled in four groups: 12 mg (n = 6), 24 mg (n = 7), 48 mg (n = 2) or matching placebo (n = 8) with 18 immunologically evaluable. T-cell immunogenicity was assessed by IFNγ ELIspot and intracellular cytokine staining (ICS). The OPAL-HIV-Gag(c) peptides were antigenic in vitro in 17/17 subjects. After vaccination with OPAL-HIV-Gag(c), 1/6 subjects at 12 mg and 1/6 subjects at 24 mg dose groups had a 2- and 3-fold increase in ELIspot magnitudes from baseline, respectively, of Gag-specific CD8+ T-cells at week 14, compared to 0/6 subjects in the placebo group. No Gag-specific CD4+ T-cell responses or overall change in Rev, Nef, Tat and CMV specific responses were detected. Marked, transient and self-limiting lymphopenia was observed immediately post-vaccination (4 hours) in OPAL-HIV-Gag(c) but not in placebo recipients, with median fall from 1.72 to 0.67 million lymphocytes/mL for active groups (P<0.001), compared to post-placebo from 1.70 to 1.56 lymphocytes/ml (P = 0.16). Despite strong immunogenicity observed in several Macaca nemestrina studies using this approach, OPAL-HIV-Gag(c) was not significantly immunogenic in humans and improved methods of generating high-frequency Gag-specific T-cell responses are required. ClinicalTrials.gov, Registry number: NCT01123915, URL trial registry database: http://www.clinicaltrials.gov/ct2/results?term=OPAL-HIV-1001&Search=Search.PLoS ONE 01/2013; 8(10):e74389. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Preclinical studies of overlapping 15mer peptides, spanning SIV, SHIV or HIV, pulsed on autologous PBMC ex vivo have demonstrated high level, virus-specific T cell responses and viral suppression in non-human primates (NHP). Opal-HIV-Gag(c) consists of 120 synthetic 15mer peptides spanning Clade C, consensus Gag, manufactured to current good manufacturing practice; having been evaluated in a good laboratory practice toxicology study in Macaca mulatta. We evaluated the safety and preliminary immunogenicity of such peptides administered intravenously after short-duration ex vivo incubation, to HIV-positive adults on suppressive antiretroviral therapy. A first-in-human, placebo-controlled, double-blind, dose escalation study was conducted. Twenty-three patients with virus suppressed by antiretroviral therapy were enrolled in four groups 12 mg (n = 6), 24 mg (n = 6), 48 mg (n = 2) or matching placebo (n = 8). Treatment was administered intravenously after bedside enrichment of 120 mL whole blood for white cells using a closed system (Sepax S-100 device), with ex vivo peptide admixture (or diluent alone) and 37°C incubation for one hour prior to reinfusion. Patients received 4 administrations at monthly intervals followed by a 12-week observation post-treatment. Opal-HIV-Gag(c) was reasonably tolerated at doses of 12 and 24 mg. There was an increased incidence of temporally associated pyrexia, chills, and transient/self-limiting lymphopenia in Opal-HIV-Gag(c) recipients compared to placebo. The study was terminated early, after two patients were recruited to the 48 mg cohort; a serious adverse event of hypotension, tachycardia secondary to diarrhoea occurred following a single product administration. An infectious cause for the event could not be identified, leaving the possibility of immunologically mediated product reaction. A serious, potentially life-threatening event of hypotension led to early, precautionary termination of the study. In the absence of a clearly defined mechanism or ability to predict such occurrence, further development of Opal-HIV-Gag(c) will not be undertaken in the current form. ClinicalTrials.gov NCT01123915; EudraCT: 2008-005142-23.PLoS ONE 09/2013; 8(9):e73765. · 3.53 Impact Factor
Control of Viremia and Prevention of AIDS following
Immunotherapy of SIV-Infected Macaques with Peptide-
Robert De Rose1, Caroline S. Fernandez1, Miranda Z. Smith1, C. Jane Batten1, Sheilajen Alca ˆntara1,
Vivienne Peut1, Erik Rollman1, Liyen Loh1, Rosemarie D. Mason1, Kim Wilson2, Matthew G. Law3,
Amanda J. Handley1,4¤, Stephen J. Kent1*
1Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia, 2National Serology Reference Laboratory, Fitzroy, Victoria,
Australia, 3National Centre for HIV Epidemiology and Clinical Research, University of New South Wales, Sydney, New South Wales, Australia, 4Opal Therapeutics Pty Ltd,
Melbourne, Victoria, Australia
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 a
booster 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 in
immunized 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 in
comparison 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, Alca ˆntara S, et al. (2008) Control of Viremia and Prevention of AIDS following Immunotherapy of SIV-
Infected 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: ? 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 Research
Council grants 299907, 251653, 454553, 359281. The funders played no role in the design and conduct of the study, nor in the collection, analysis and
interpretation 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: email@example.com
¤ Current address: Medicines Development Ltd, Melbourne, Victoria, Australia
Several attempts at immunotherapy of HIV using more
conventional 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 to
deliver HIV immunotherapies has shown strong immunogenicity
efficacy in macaques and pilot humans studies but is limited to
highly specialized facilities [5,6,7]. A simple intermittent immu-
notherapy that reduces the need for long-term antiretroviral
therapy (ART) would be a quantum advance in treating HIV.
We recently reported the robust T-cell immunogenicity of
treating unfractionated whole blood or peripheral blood mono-
nuclear cells (PBMC) with overlapping peptides of SIV, HIV-1 or
hepatitis C virus in outbred pigtail monkeys [8,9]. We termed this
Autologous ceLls). This technique is attractive since there is no
prolonged ex vivo culture of antigen-presenting cells, robust CD4
and CD8 T-cell responses to both structural and regulatory
proteins can be induced, and peptide antigens are simple to
manufacture 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 highlight
the 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 as
effective as more broadly targeting all 9 SIV proteins.
Materials and Methods
Juvenile pigtail macaques (Macaca nemestrina) free from Simian
retrovirus type D were studied in protocols approved by
PLoS Pathogens | www.plospathogens.org1May 2008 | Volume 4 | Issue 5 | e1000055
institutional animal ethics committees and cared for in accordance
with 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 the
presence of Mane-A*10 confirmed by sequence specific primer
PCR as described [12,13]. 36 macaques were injected intrave-
nously 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. Ran-
domization was stratified for peak SIV viral load at week 2, weight,
gender and the MHC I gene Mane-A*10 (which is known to
enhance immune control of SIV) . Animals received
subcutaneous injections of dual anti-retroviral therapy with
tenofovir 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 from
weeks 6–10. This dual ART controls viremia in the majority of
SIV-infected macaques [16,17,18,19,20].
Two animal groups (OPAL-Gag and OPAL-All) were immu-
nized with OPAL immunotherapy using PBMC as previously
described . Briefly, peripheral blood mononuclear cells (PBMC)
were isolated over Ficoll-paque from 18 ml of blood (anticoagu-
lated 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 SIVmac239proteins (Gag, Pol, Env, Nef, Vif, Tat, Rev,
Vpr, Vpx) were added at 10 mg/ml of each peptide within the
pool. 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, 6448-
50, 6407, 8762, 6205). To pool the peptides, each 1 mg vial of
lyophilised 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 held
for 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 were
adapted from effective stimulation of T cell responses in vitro.
Control macaques did not receive vaccine treatment. This was
done since (a) we had not previously observed any significant VL
changes with non HIV/SIV peptide sets ([8,9] and unpublished
data), (b) reinfusion of blood cells pulsed with irrelevant sets of
peptides would result in some level of immune activation and drive
higher viral loads in controls, artificially magnifying any reductions
in the active treatment groups, (c) reinfusion of control peptide
pulsed cells might have obscured any unexpected safety problems
of the procedure.
SIV-specific CD4 and CD8 T-cell immune responses were
analysed by expression of intracellular IFNc as previously
described . 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/Pharmin-
gen 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) and
the 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-specific
gated lymphocytes expressing IFNc was assessed in both
CD3+CD4+and CD3+CD8+lymphocyte subsets. Responses to
the immunodominant SIV Gag CD8 T-cell epitope KP9 in Mane-
A*10+ animals were assessed by a Mane-A*10/KP9 tetramer as
described . Total peripheral CD4 T-cells were measured as a
proportion of lymphocytes by flow cytometry on fresh blood.
Plasma SIV RNA was quantitated by real time PCR on 140 ml
of plasma at the University of Melbourne (lower limit of
quantitation 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 of
quantitation 1.5 log10copies/ml) as previously described .
The primary endpoint was the reduction in plasma SIV RNA in
OPAL-immunized animals compared to controls by time-
weighted area-under-the-curve (TWAUC) for 10 weeks following
withdrawal of ART (i.e. samples from weeks 12 to 20). This
summary statistical approach is recommended for studies such as
these involving serial measurements . We compared both
active treatment groups (OPAL-Gag and OPAL-All) to controls
separately and together. The primary analysis was restricted to
animals that controlled viremia on the ART at week 10 (VL,3.1
log10copies/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) and
Mane-A*10 status. Group comparisons used two-sample t-tests for
continuous data, and Fisher’s exact test for binary data. Survival
analyses utilised Cox-regression analyses.
Prior to initiating the study, we estimated the standard deviation
of the return of VL after treatment interruption would be
approximately 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
Effective immunotherapies for HIV are needed. We
assessed a simple technique, reinfusion of fresh blood
cells incubating with overlapping SIV peptides (Overlap-
ping Peptide-pulsed Autologous ceLls, OPAL), in 36
randomly allocated SIV-infected monkeys. We analyzed
this therapy for the stimulation of immunity, control of
virus levels, and prevention of AIDS. The OPAL immuno-
therapy was safe and stimulated remarkable levels of T-cell
immunity. Levels of virus in vaccinated monkeys were 10-
fold 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 a
promising immunotherapy technique. Trials in HIV-infect-
ed humans of OPAL therapy are planned.
OPAL Immunotherapy for SIV
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infection. A 10 control vs 10 active treatment comparison yields
80% power (p=0.05) to detect a 1.0 log10difference 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
This 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
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 ), weight and
gender. Macaques were immunized 4 times under the cover of
antiretroviral 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 un-
immunized. The macaques were initially followed for 26 weeks
after ceasing ART on week 10.
All 36 macaques became infected following SIVmac251exposure
and had a mean peak VL of 7.1 log10copies/ml (Table S1). Prior
to vaccination, 4 animals died during acute SIV infection with
diarrhoea, 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).
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% and
1.9% 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 OPAL-
All group and 0.15% and 0.29% in controls (Fig. 1A, B). The Gag-
specific T cells in the OPAL-All immunized animals, but not
control or OPAL-Gag only immunized animals, also had elevated
T-cell responses to all other SIV proteins. Mean Env, Pol and
combined regulatory protein-specific CD4/CD8 responses were
2.5%/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 to
Gag (Fig. 1E). Thus, although a larger number of SIV proteins
were recognized in the OPAL-All immunized animals, Gag
responses were reduced in comparison to only immunizing with
Although the short linear peptides were primarily used to
induce 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).
Virologic outcome following initial vaccinations
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 (26
animals). 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) and
higher 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 log10copies/ml (p=0.028,
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) . The University of Melbourne and NCI
assays were tightly correlated (r=0.97, p,0.001) and showed an
almost identical mean reduction in viremia in vaccinees compared
to controls at this time (0.82 vs 0.88 log10copies/ml respectively).
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 identical
procedure (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 to
the 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 enhancement
of 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 ‘‘last
OPAL Immunotherapy for SIV
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Figure 1. T-cell immunogenicity of OPAL vaccination. SIV Gag-specific CD4 (A) and CD8 (B) T-cells expressing IFNc were studied over time by
intracellular 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 SIVmac251infection) 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 of
antiretroviral 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,
sided 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.
OPAL Immunotherapy for SIV
PLoS Pathogens | www.plospathogens.org4May 2008 | Volume 4 | Issue 5 | e1000055
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
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
Figure 2. Non-Gag T cell immunogenicity of OPAL Vaccination. SIV-specific CD4 and CD8 T-cells expressing IFNc were studied over time by
intracellular 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.
OPAL Immunotherapy for SIV
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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.
OPAL Immunotherapy for SIV
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that did not control viremia on ART required euthanasia. Of the 6
euthanised 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 and
control of viremia on ART (p=0.02, Table 1).
In summary, OPAL immunotherapy, either using overlap-
ping 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 unvacci-
nated controls. The virologic efficacy in OPAL-immunized
macaques was durable for 12 months after ART cessation. Our
findings on OPAL immunotherapy were observed despite the
virulent SIVmac251-pigtail model studied  and provide strong
proof-of-principle for the promise of this immunotherapy
The OPAL immunotherapy approach is simpler than many
other 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 macaque
studies [17,27], although such approaches have not yet been
translated 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, a
technique that will be more widely applicable ( and unpublished
This is one of the largest therapeutic SIV vaccine studies yet
reported. Although it may have been ideal to have studied
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).
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irrelevant peptide-pulsed autologous cells as an additional control
group, we were concerned that this may have magnified the
therapeutic effect or obscured any safety concerns. In the end, the
vaccination process was both safe and effective.
How well the findings on OPAL immunotherapy translate to
humans with acute HIV-1 infection will be determined by clinical
trials. Virus-specific CD4 T cells are typically very weak in HIV-
infected humans or SIV-infected macaques; dramatic enhance-
ment of these cells were induced by OPAL immunotherapy and
this may underlie its efficacy . 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 can
also 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 log10reduction in VL would result in a substantial
delay in progressive HIV disease in humans and allow a
reasonable time period without the requirement to reintroduce
ART  if these findings are confirmed in human trials. Both
the control and vaccinated macaques were treated with ART
early in this study (3 weeks after infection), which alone can be
associated with a transiently improved outcome in humans .
None-the-less, a massive loss of CD4+ T cells in the gut occurs
within 2 weeks of infection . Although it may be challenging
to identify humans within 3 weeks of infection, this is when HIV-
1 subjects typically present with acute infection. The durable
control of viremia exhibited by the vaccinated animals is
interesting and consistent with other recent macaque studies
, 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, non-
significant, 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 and
benefit of ART cover during boosting immunizations with OPAL
Control of viremia was similar for the OPAL-Gag and OPAL-
All groups. Gag-specific CD4 and CD8 T-cell responses in OPAL-
Gag 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 the
development of Gag-specific T-cell responses . A large human
cohort study demonstrated Gag-specific T-cell responses were the
most effective in controlling HIV viremia . Useful subdom-
inant 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 of
this flexible vaccine technology. Therapeutic HIV vaccines may
not need to aim for maximally broad multi-protein HIV-specific
OPAL immunotherapy with Gag peptides is proceeding into
initial trials in HIV-infected humans. Additional peptides can
readily be added into standard consensus strains mixes to cover
common strain or subtype variations between strains with this
technology . Additional technologies such as toggling variable
amino acids peptides may provide further T cell immunogenicity
with this general technology . Immunotherapy with peptides
delivered onto fresh blood may have potential applicability for
other chronic viral diseases such as hepatitis C virus infection and
some cancers such as melanoma .
Table 1. Statistical analyses of VL and Survival.
Reduction in VL
10 weeks off ARTb
2 sided t-test
Reduction in VL
6 months off ART
2 sided t-test
Reduction in VL
1 year off ARTc
2 sided t-test
Survival 1 year
off ART (p-value)d
VL undetectable at
OPAL- Gag+All vs
16 vs 10
Individual vaccine arms OPAL-All vs controls
8 vs 10
OPAL-Gag vs controls
8 vs 10
All animals, adjusted for
Mane-A*10 status and VL
at week 10 (n=32)
21 vs 11
Individual vaccine arms OPAL-All vs controls
11 vs 11
OPAL-Gag vs controls
10 vs 11
a6 of the 32 animals failed to control viremia on ART.
bVL values reductions are log10copies/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 the
end of the period in parentheses.
c12 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.
OPAL Immunotherapy for SIV
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Found at: doi:10.1371/journal.ppat.1000055.s001 (0.08 MB PDF)
Viral load data
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 SIVmac251stock,
M. Miller for the antiretrovirals, and M. Piatak and J. Lifson for viral load
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.
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