Trivalent adenovirus type 5 HIV recombinant vaccine primes for modest cytotoxic capacity that is greatest in humans with protective HLA class I alleles.
ABSTRACT If future HIV vaccine design strategies are to succeed, improved understanding of the mechanisms underlying protection from infection or immune control over HIV replication remains essential. Increased cytotoxic capacity of HIV-specific CD8+ T-cells associated with efficient elimination of HIV-infected CD4+ T-cell targets has been shown to distinguish long-term nonprogressors (LTNP), patients with durable control over HIV replication, from those experiencing progressive disease. Here, measurements of granzyme B target cell activity and HIV-1-infected CD4+ T-cell elimination were applied for the first time to identify antiviral activities in recipients of a replication incompetent adenovirus serotype 5 (Ad5) HIV-1 recombinant vaccine and were compared with HIV-negative individuals and chronically infected patients, including a group of LTNP. We observed readily detectable HIV-specific CD8+ T-cell recall cytotoxic responses in vaccinees at a median of 331 days following the last immunization. The magnitude of these responses was not related to the number of vaccinations, nor did it correlate with the percentages of cytokine-secreting T-cells determined by ICS assays. Although the recall cytotoxic capacity of the CD8+ T-cells of the vaccinee group was significantly less than that of LTNP and overlapped with that of progressors, we observed significantly higher cytotoxic responses in vaccine recipients carrying the HLA class I alleles B*27, B*57 or B*58, which have been associated with immune control over HIV replication in chronic infection. These findings suggest protective HLA class I alleles might lead to better outcomes in both chronic infection and following immunization due to more efficient priming of HIV-specific CD8+ T-cell cytotoxic responses.
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ABSTRACT: Interrogating immune correlates of infection risk for efficacious and non-efficacious HIV-1 vaccine clinical trials have provided hypotheses regarding the mechanisms of induction of protective immunity to HIV-1. To date, there have been six HIV-1 vaccine efficacy trials (VAX003, Vaxgen, Inc., San Francisco, CA, USA), VAX004 (Vaxgen, Inc.), HIV-1 Vaccine Trials Network (HVTN) 502 (Step), HVTN 503 (Phambili), RV144 (sponsored by the U.S. Military HIV Research Program, MHRP) and HVTN 505). Cellular, humoral, host genetic and virus sieve analyses of these human clinical trials each can provide information that may point to potentially protective mechanisms for vaccine-induced immunity. Critical to staying on the path toward development of an efficacious vaccine is utilizing information from previous human and non-human primate studies in concert with new discoveries of basic HIV-1 host-virus interactions. One way that past discoveries from correlate analyses can lead to novel inventions or new pathways toward vaccine efficacy is to examine the intersections where different components of the correlate analyses overlap (e.g., virus sieve analysis combined with humoral correlates) that can point to mechanistic hypotheses. Additionally, differences in durability among vaccine-induced T- and B-cell responses indicate that time post-vaccination is an important variable. Thus, understanding the nature of protective responses, the degree to which such responses have, or have not, as yet, been induced by previous vaccine trials and the design of strategies to induce durable T- and B-cell responses are critical to the development of a protective HIV-1 vaccine.Vaccines. 03/2014; 2(1):15-35.
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ABSTRACT: The extraordinary variability of HIV-1 poses a major obstacle to vaccine development. The effectiveness of a vaccine is likely to vary dramatically in different populations infected with different HIV-1 subtypes, unless innovative vaccine immunogens are developed to protect against the range of HIV-1 diversity. Immunogen design for stimulating neutralizing antibody responses focuses on "breadth" - the targeting of a handful of highly conserved neutralizing determinants on the HIV-1 Envelope protein that can recognize the majority of viruses across all HIV-1 subtypes. An effective vaccine will likely require the generation of both broadly cross-neutralizing antibodies and non-neutralizing antibodies, as well as broadly cross-reactive T cells. Several approaches have been taken to design such broadly-reactive and cross-protective T cell immunogens. Artificial sequences have been designed that reduce the genetic distance between a vaccine strain and contemporary circulating viruses; "mosaic" immunogens extend this concept to contain multiple potential T cell epitope (PTE) variants; and further efforts attempt to focus T cell immunity on highly conserved regions of the HIV-1 genome. Thus far, a number of pre-clinical and early clinical studies have been performed assessing these new immunogens. In this review, the potential use of these new immunogens is explored.Viruses 10/2014; 6(10):3968-3990. · 3.28 Impact Factor
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ABSTRACT: The contribution of host T-cell immunity and HLA class I alleles to control of HIV replication in natural infection is widely recognized. We assessed whether vaccine-induced T-cell immunity, or expression of certain HLA alleles, impacted HIV control post-infection in the Step MRKAd5/HIV-1 gag/pol/nef Study (ClinicalTrials.gov Identifier: NCT00095576). Vaccine-induced T cells were associated with reduced plasma viremia, with subjects targeting ≥3 Gag peptides presenting with half-log lower mean viral load than subjects without Gag responses. This effect was stronger in participants infected proximal to vaccination, and was independent of our observed association of HLA-B*27, -B*57 and -B*58:01 alleles with lower HIV viremia. These findings support the ability of vaccine-induced T-cell responses to influence post-infection outcome, and provide a rationale for the generation of T-cell responses by vaccination to reduce viremia if protection from acquisition is not achieved.The Journal of Infectious Diseases 07/2013; · 5.85 Impact Factor
Trivalent Adenovirus Type 5 HIV Recombinant Vaccine
Primes for Modest Cytotoxic Capacity That Is Greatest in
Humans with Protective HLA Class I Alleles
Stephen A. Migueles1, Julia E. Rood1, Amy M. Berkley1, Tiffany Guo1, Daniel Mendoza1, Andy
Patamawenu1, Claire W. Hallahan3, Nancy A. Cogliano1, Nicole Frahm2, Ann Duerr2, M. Juliana
McElrath2, Mark Connors1*
1Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America,
2Vaccine and Infectious Disease Division and the HIV Vaccine Trials Network, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America,
3Biostatistics Research Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
If future HIV vaccine design strategies are to succeed, improved understanding of the mechanisms underlying protection
from infection or immune control over HIV replication remains essential. Increased cytotoxic capacity of HIV-specific CD8+T-
cells associated with efficient elimination of HIV-infected CD4+T-cell targets has been shown to distinguish long-term
nonprogressors (LTNP), patients with durable control over HIV replication, from those experiencing progressive disease.
Here, measurements of granzyme B target cell activity and HIV-1-infected CD4+T-cell elimination were applied for the first
time to identify antiviral activities in recipients of a replication incompetent adenovirus serotype 5 (Ad5) HIV-1 recombinant
vaccine and were compared with HIV-negative individuals and chronically infected patients, including a group of LTNP. We
observed readily detectable HIV-specific CD8+T-cell recall cytotoxic responses in vaccinees at a median of 331 days
following the last immunization. The magnitude of these responses was not related to the number of vaccinations, nor did it
correlate with the percentages of cytokine-secreting T-cells determined by ICS assays. Although the recall cytotoxic capacity
of the CD8+T-cells of the vaccinee group was significantly less than that of LTNP and overlapped with that of progressors,
we observed significantly higher cytotoxic responses in vaccine recipients carrying the HLA class I alleles B*27, B*57 or B*58,
which have been associated with immune control over HIV replication in chronic infection. These findings suggest
protective HLA class I alleles might lead to better outcomes in both chronic infection and following immunization due to
more efficient priming of HIV-specific CD8+T-cell cytotoxic responses.
Citation: Migueles SA, Rood JE, Berkley AM, Guo T, Mendoza D, et al. (2011) Trivalent Adenovirus Type 5 HIV Recombinant Vaccine Primes for Modest Cytotoxic
Capacity That Is Greatest in Humans with Protective HLA Class I Alleles. PLoS Pathog 7(2): e1002002. doi:10.1371/journal.ppat.1002002
Editor: Ronald C. Desrosiers, Harvard University, United States of America
Received September 24, 2010; Accepted December 21, 2010; Published February 24, 2011
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This research was supported in part by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases. The HVTN
Laboratory Program (NIH U01 AI068618), the Seattle Vaccine Unit (NIH U01 AI069481) and HVTN Core Leadership (U01 AI068614) provided additional support. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Understanding the fundamental basis of immunologic control of
HIV remains an enormous challenge in the development of
efficacious HIV vaccines and immunotherapies. Some important
clues have emerged from studies of rare patients with natural
immune control over HIV referred to as long-term nonprogressors
(LTNP), HIV controllers, elite suppressors or elite controllers who
contain HIV replication for many years to less than 50 copies/mL
plasma without antiretroviral therapy (ART) (reviewed in ).
Several lines of evidence suggest that HIV-specific CD8+T-cell
responses are responsible for mediating immune control in these
individuals. Among these are strong, consistent associations
between nonprogressive infection and particular HLA class I
alleles like B*57 [2–8]. In B*57+LTNP, this genetic association is
paralleled by functional data demonstrating an overwhelming
immunodominance of HLA B57-restricted, HIV-specific CD8+T-
cells [2,9,10]. Similar observations between protective MHC
alleles, like Mamu B*08 and B*17, and prolonged restriction of
SIV replication have been made in the rhesus macaque model of
SIV infection [11–13]. Greater insight into the mechanisms
underlying these associations, which are among the strongest
observed in human diseases as determined by a number of
approaches, will certainly enhance our understanding of the
parameters necessary for the induction and/or maintenance of
immune-mediated control of HIV infection.
Recently, several important advances have been made in
understanding the mechanism of immunologic control of HIV in
humans. It has been known for some time that patients with
immunologic control are not distinguished by greater frequencies
or breadth of HIV-specific CD8+T-cells or by the particular
specificities that are targeted [2,14–16]. These observations have
suggested that the CD8+T-cells of LTNP are not differentiated
from those of progressors on the basis of quantitative consider-
ations. HIV-specific CD8+T-cells of LTNP have been observed to
mediate a greater number of functions based upon cytokine and
chemokine secretion compared to progressors, although there is
considerable overlap between these patient groups [17–19]. Most
PLoS Pathogens | www.plospathogens.org1February 2011 | Volume 7 | Issue 2 | e1002002
notably, the CD8+T-cells of LTNP have been distinguished from
those of progressors based upon their ability to suppress HIV
replication in vitro or in humanized mice [5,20]. The mechanism
underlying this suppressive capacity is the greater ability of LTNP
CD8+T-cells to expand and produce the pore-forming molecule
perforin, which is necessary for granule-exocytosis mediated
cytotoxicity [8,19,21]. HIV-specific CD8+T-cells of LTNP possess
extraordinary cytotoxic capacity against primary HIV-infected
target cells on a per-cell basis measured by granzyme B target cell
activity and infected CD4+T-cell elimination [8,19]. This is a
function that clearly differentiates these individuals from untreated
or treated patients without immune-mediated control of HIV.
High levels of cytotoxic capacity are not recovered when the level
of antigen is reduced through antiretroviral therapy .
Although this function clearly distinguishes those with immuno-
logic control in the setting of chronic infection, it may not
necessarily be the operative mechanism in control induced by an
HIV vaccine. Thus far, direct measurements of recall cytotoxic
capacity assessed by granzyme B target cell activity and infected
CD4+T-cell elimination have not been applied to recipients of
Although a number of vaccine candidates under development
aim to induce cellular immune responses directed against HIV,
those among the most immunogenic based upon pre-clinical and
phase I clinical studies have been the replication incompetent
adenovirus serotype 5 (Ad5) HIV-1 recombinant vaccines [22–25].
An efficacy trial employing such a vaccine was the Step study, a
phase IIB test-of-concept trial involving 3,000 HIV-negative
individuals at high risk of HIV infection [26,27]. After one year
of follow up, HIV RNA levels were comparable among those who
became infected regardless of immunization . Initial analyses,
using a validated interferon-c ELISPOT assay and an intracellular
cytokine staining (ICS) assay, revealed no differences in vaccine-
induced HIV-specific immunity, including response rate, magni-
tude and cytokine profile, between male cases and non-cases .
These findings suggested that if vaccine-induced immunologic
control is to succeed, future candidate vaccines may need to elicit
responses of higher magnitude or different breadth or function.
In addition to these findings, some data have suggested an impact
of host HLA in response to vaccination. CD8+T-cells restricted by
protective alleles were observed to dominate the response to
vaccination with an ALVAC-HIV recombinant canarypox . In
addition, in one early analysis of the Step study, more individuals
carrying HLA class I alleles that have been associated with immune
control in the chronic phase of infection like B*57 and B*27, who
subsequently became infected with HIV, were controlling HIV
replication to low levels (Nicole Frahm, personal communication)
although, in a more recent analysis, this did not achieve statistical
significance. These data are preliminary and the numbers are small.
It is important to note that vaccinee cases were not distinguished by
quantitative measurements of their HIV-specific CD8+T-cell
response by ELISPOT (Nicole Frahm, personal communication).
In the present study, we examined the HIV-specific CD8+
T-cell cytotoxic capacity of HIV-1-uninfected recipients of the
Merck Ad5/HIV trivalent vaccine. We observed CD8+T-cells in
some vaccinees exhibiting proliferative capacity and the ability to
upregulate perforin expression that overlapped with those of
LTNP. However, the ability of these cells to kill primary
autologous HIV-infected targets was relatively low and compara-
ble to that of progressors. This low cytotoxic capacity was not
related to precursor or effector cell frequencies. Although this
cytotoxic capacity was modest in most vaccinees, the highest
responses were observed in those with the protective HLA class I
alleles B*57, B*58 and B*27. Taken together these data suggest
that the Merck Ad5/HIV trivalent vaccine induced relatively
modest cytotoxic capacity against HIV-1-infected cells in vacci-
nees. In addition, they suggest that protective HLA class I alleles
have an effect on the cytotoxic capacity induced by vaccination
that is mediated during T-cell priming.
Materials and Methods
For this study, approval to transfer samples from immunized
Human Immunodeficiency Virus (HIV)-seronegative participants
of HIV Vaccine Trials Network (HVTN) protocols 071 or 502 was
granted by the Fred Hutchinson Cancer Research Center
Institutional Review Board (IRB# 00000022). HIV-infected
subjects and non-immunized HIV-seronegative controls were
recruited from the Clinical Research Center, National Institutes
of Health (Bethesda, MD) and signed National Institute of Allergy
and Infectious Diseases Investigational Review Board (NIAID
IRB#5)-approved protocol informed consent documents. The
study was conducted according to the principles expressed in the
Declaration of Helsinki.
As part of HVTN protocols 071 or 502, 31 HIV-negative
individuals received 2 (n=19) or 3 (n=12) immunizations,
respectively, of the Merck recombinant, replication-incompetent
adenovirus serotype 5 (Ad5) HIV-1 trivalent vaccine, which was a
mixture of three E1-deleted recombinant Ad5 viruses, each
containing one of three HIV-1 inserts (gag, pol and nef), as described
previously . The vaccine was administered intramuscularly as a
1-mL injection of 1.561010adenovirus genomes. The vaccine
recipients underwent large volume venipuncture or leukapheresis a
median of 331 (range, 8–1,315) days after the last vaccination at the
site of enrollment. The first 19 samples, which were randomly
selected, contained cells from only 5 individuals with the protective
HLA class I alleles B*27, B*57 or B*58. Therefore, 12 additional
Unique HIV-infected individuals have remained healthy
with stable CD4 counts and HIV RNA levels below the
detection threshold in sensitive assays without antiretro-
viral therapy for 20 years. These nonprogressors have been
intensively studied in order to identify mechanisms that
could inform the design of an efficacious HIV/AIDS vaccine.
In addition to strong associations with certain host genes
like HLA B*57, nonprogressors are distinguished from
progressors by the superior ability of their HIV-specific
CD8+T-cells to proliferate and to efficiently kill HIV-
infected CD4+T-cell targets via perforin and granzyme B,
the major proteins contained within killing granules. Here,
for the first time, we apply sensitive measurements of
CD8+T-cell proliferation and perforin expression, gran-
zyme B target cell activity and infected CD4+T-cell
elimination to samples derived from recipients of the
Merck adenovirus serotype 5-HIV vaccine. We demonstrate
readily detectable CD8+T-cell-mediated killing in these
vaccinees. Although the killing responses were less than
those of nonprogressors, vaccinees expressing the protec-
tive HLA alleles B*27, B*57 or B*58 exhibited greater killing
than those not possessing these alleles. These findings
suggest protective HLA alleles lead to better outcomes in
both chronicinfection and
through early interactions that induce superior antiviral
CD8+T-cell killing responses.
HIV-Specific Cytotoxic Capacity in Vaccinees
PLoS Pathogens | www.plospathogens.org2February 2011 | Volume 7 | Issue 2 | e1002002
samples, including cells from 6 patients with protective HLA class I
alleles, were provided. In total, 11 vaccinees carried one of these
protective HLA class I B alleles versus 20 vaccinees who did not
(Table S1). Investigators performing the studies were blinded to the
HLA haplotype of the vaccinees. HLA class I typing was performed
by a high-throughput sequencing based PCR method as described
HIV infection was documented by HIV-1/2 immunoassay in
LTNP, viremic progressors and antiretroviral therapy (ART)-treated
progressors who were defined as described previously (Table S2)
[8,19]. ART-treated progressors (Rx,50) received continuous ART
and had HIV RNA levels suppressed to ,50 copies/ml for a median
of 8 (range 5–9) years. Median CD4+T-cell counts for LTNP,
viremic progressors and Rx,50 were 955 (range 664–1,362), 449
(range 238–739) and 692 (range 408–720) cells/ml, respectively.
Since HIV-specific CD8+T-cell functionality with respect to
proliferative and cytotoxic capacities has been shown to be similar
between viremic progressors and Rx,50 [8,19], these subgroups
were combined into a single progressor group in the statistical
analyses. Peripheral blood mononuclear cells (PBMC) were obtained
as described previously . HLA class I/II typing was performed by
sequence-specific hybridization as described previously .
HIVSF162-infected autologous CD4+T-cell targets
CD4+T-cells were positively selected from cryopreserved
PBMC derived from vaccinees and chronically infected patients
by magnetic automated cell sorting (AutoMACS, Miltenyi Biotec,
Germany) and polyclonally stimulated with medium containing
anti-CD3 (Orthoclone OKT3, 1 mg/ml; Ortho Biotech, Bridge-
water, NJ), anti-CD28 (1 mg/ml, BD Biosciences) and human IL-2
(40 IU/ml, Roche Diagnostics, Indianapolis, IN) prior to infection
as previously described . CD4+lymphoblasts were infected
over 24–36 hours as recently described [8,19,30]. The percent
infection of CD4+T-cell targets was confirmed in all cases by
intracellular HIV-1 Gag p24 expression using flow cytometry and
was similar among LTNP (median 59.9%), progressors (56.9%),
HIV-negative controls (60.5%) and vaccine recipients (69.4%).
Granzyme B cytotoxicity assay and infected CD4+T-cell
Day 6 effector cells (PBMC incubated with infected targets for 6
days) were labeled with immuno-magnetic beads (CD8+T-cell
Isolation Kit II, Miltenyi Biotec) prior to negative selection of
CD8+T-cells by magnetic automated cell sorting as described
previously . Cytotoxic responses were measured against LIVE/
DEAD Fixable Violet Stain (Molecular Probes, Invitrogen
Detection Technologies, Eugene, OR, USA)-labeled HIVSF162-
infected and uninfected autologous CD4+T-cell targets in assays
examining GrB target cell activity and infected CD4+T-cell
elimination (ICE) as recently reported [8,19]. To assess per-cell
cytotoxic capacity, ICE responses were measured at a standard
E:T ratio of 25:1 (total day 6 CD8+T-cells to total CD4+T-cell
targets) for all individuals, and additionally at an E:T ratio of 50:1
in 20 vaccinees from whom greater numbers of PBMC were
available. These responses were then plotted against the true E:T
ratios, which were determined by measurements of IFN-c-
secreting CD8+T-cells and p24-expressing target cells (sum of
CD4-p24+and CD4+p24+cells in plots containing only infected
targets), respectively, as previously described [8,19].
CFSE proliferation assays
PBMC were labeled with 5,6-carboxyfluorescein diacetate,
succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) and
incubated with medium, anti-CD3 (Orthoclone OKT3, 1 mg/ml)
and anti-CD28 (1 mg/ml) antibodies or uninfected or HIVSF162-
infected autologous CD4+T-cell targets in 96-well, deep-well
culture plates (PGC Scientifics, Frederick, MD) at a density of 106
PBMC/well/ml for 6 days as previously described .
CD8+T-cell stimulation assays for intracellular protein
In experiments using CD4+T-cell targets to measure the total
frequency of virus-specific CD8+T-cells in cytotoxicity experi-
ments, negatively selected CD8+T-cells (incubated with HIVSF162-
infected targets for 6 days) were co-incubated with uninfected or
HIVSF162-infected autologous CD4+T-cell targets for 6 hours
prior to fixation, permeabilization and intracellular IFN-c staining
as described previously [8,19].
In flow cytometric intracellular cytokine staining (ICS) assays
employed to enumerate HIV-specific T-cells in vaccine recipients,
thawed PBMC were cultured overnight and then stimulated for
6 hours with HIV-1 Nef, Gag or Pol peptide pools that span the
sequence encoded by the HIV-1 gene inserts in the vaccine in
order to detect IFN-c and IL-2 production by CD8+and CD4+T-
cells, as described previously . Pools of potential T-cell epitope
(PTE) 15-mer peptides were used in the 071 study and pools of
15-mer peptides overlapping in sequence by 11 amino acids were
used in the 502 study. The criteria for positive and negative
responses were defined previously .
Multiparameter flow cytometry was performed according to
standard protocols. Surface and/or intracellular staining was done
using the following antibodies from BD Biosciences, unless
otherwise noted: fluorescein isothiocyanate (FITC)-conjugated
anti-CD3; peridinine chlorophyll protein (PerCP)-conjugated anti-
CD3 and anti-CD4; allophycocyanin (APC)-conjugated anti-CD4,
anti-CD8 and anti-IFN-c; APC H7-conjugated anti-CD3; phyco-
erythrin (PE)-conjugated anti-CD8; Pacific Blue-conjugated anti-
perforin and RDI-conjugated anti-p24 antibodies (Kc57, Beckman
Coulter, Inc., Fullerton, CA). Unless otherwise specified, all staining
was performed at 4uC for 30 minutes. In cytotoxicity experiments,
gates were drawn on labeled CD4+T-cell targets and 5,000-8,000
events were collected. Samples were analyzed on a FACSAria
multi-laser cytometer (Becton-Dickinson) with FACSDiva software.
Color compensations were performed using single-stained samples
for each of the fluorochromes used. Data were analyzed using
FlowJo software (TreeStar, San Carlos, CA).
The Wilcoxon signed rank test was used to compare paired data.
Independent groups were compared by the Wilcoxon two-sample
test. Correlation was determined by the Spearman rank method.
The Bonferroni method was used to adjust p values for multiple
testing. Regression analysis, analysis of covariance and repeated
measures were used to quantify the differences in ICE among
ranges of logged E:T ratios, at the median logged E:T ratio of the
combined independent groups and at the logged E:T ratio of 5:1.
HIV-specific CD8+T-cell cytotoxic responses induced by
an Ad5/HIV vaccine are similar to those of progressors
We have previously observed large differences in cytotoxic
capacity between LTNP and viremic or ART-suppressed
HIV-Specific Cytotoxic Capacity in Vaccinees
PLoS Pathogens | www.plospathogens.org3 February 2011 | Volume 7 | Issue 2 | e1002002
progressors, however, these measurements have not been applied
to vaccinees. In their current form, these assays require large
numbers of PBMC. For this reason, cells from participants in two
trials (HVTN 071 and 502) in which vaccinees were leukapheresed
or gave large blood volumes after either 2 or 3 doses, respectively,
of the Merck Ad5/HIV trivalent vaccine were used. We have
observed in prior work that although some differences in the
cytotoxic capacity of unstimulated HIV-specific CD8+T-cells were
detectable between groups of chronically infected patients, the
differences were largest in a recall response after a 6-day
incubation with infected CD4+
represents the time necessary for upregulation of perforin under
conditions of low levels of antigen such as would be expected in
both LTNP and vaccinees. HIV-specific CD8+T-cell cytotoxic
responses measured by granzyme (Gr) B target cell activity and
infected CD4+T-cell elimination (ICE) were readily detectable in
vaccine recipients (Table S1, Figure S1, Figure 1). The recall
cytotoxic responses mediated by the cells of vaccinees (median
GrB activity 15.6%, range 3-37.7%; median ICE 32.6%, range
5.8-61.5%) were significantly higher than those observed in HIV-
negative controls (median GrB activity 1.7%, range 0-7.46%,
p,0.001; median ICE 0.29%, range 0-3.3%, p,0.001; Figure 1A,
B). In comparisons with chronically HIV-infected patients (Table
S2), cytotoxic responses of vaccinees were significantly lower than,
and completely non-overlapping with, those of LTNP (median
T-cells [8,19]. This likely
GrB activity 50.7%, range 42.9-63%, p,0.001; median ICE
82.5%, range 75.2-86.6%, p,0.001; Figure 1A, B). Their
responses were, however, comparable to those of progressors
(median GrB activity 16.55%, range 3.03-39%, p.0.5; median
ICE 37.35%, range 3.9-56%, p.0.5; Figure 1A, B). HIV-specific
CD8+T-cell cytotoxic responses measured by GrB or ICE were
strongly, directly correlated with each other (R=0.92, p,0.001),
as observed previously in chronically infected patients (Figure 1C)
Numbers of immunizations and time from last
vaccination did not correlate with the magnitude of
HIV-specific CD8+T-cell cytotoxic responses induced by
an Ad5/HIV vaccine
The magnitude of vaccine-induced responses may be related to
the potency and frequency of immunization, which may also
influence durability of the response. In this study, the cytotoxic
capacity of HIV-specific CD8+T-cells derived from Ad5/HIV
vaccine recipients was analyzed in the context of the number of
immunizations and time since last immunization. No significant
differences were observed in the magnitude of the CD8+T-cell
cytotoxic responses between individuals who had received 2 versus
3 immunizations (median ICE 37.2% versus 32.45%, respectively,
p.0.5; Figure 2A). In addition, there was no correlation between
the magnitude of ICE and the duration of time following the last
Figure 1. HIV-specific CD8+ +T-cell cytotoxic responses induced by an Ad5/HIV vaccine were similar to those of progressors.
(A, B) Summary data of the total cytotoxic response measured by GrB activity (circles, A) or infected CD4+T-cell elimination (ICE) (diamonds, B) with
day 6 (D#6) CD8+T-cells derived from LTNP (red symbols, n=7), progressors (blue symbols, n=10), HIV-negative individuals (black symbols, n=10)
and Ad5/HIV vaccinees (green symbols, n=31). Data are representative of at least two experiments. Comparisons were made using the Wilcoxon
two-sample test. Horizontal lines indicate median values. Only P values referring to comparisons between the responses of vaccinees and the other
groups are shown. (C) Using D#6 CD8+T-cells, GrB target cell activity correlates directly with ICE (n=58). Correlation was determined by the
Spearman rank method.
HIV-Specific Cytotoxic Capacity in Vaccinees
PLoS Pathogens | www.plospathogens.org4 February 2011 | Volume 7 | Issue 2 | e1002002
immunization (p.0.5; Figure 2B). Some of the highest responses
were observed 28 months after the last immunization. These
results further support the relative immunogenicity of this vaccine
construct, which induced significant cytotoxic responses following
2 doses, and are consistent with prior work showing little
additional increase in the frequencies of HIV-specific CD8+
T-cells with a third vaccine dose . These data also support the
induction of memory HIV-specific CD8+T-cells that persist and
have retained expansion potential.
Per-cell cytotoxic capacity of HIV-specific CD8+T-cells
induced by an Ad5/HIV vaccine was comparable to that
The differences in the magnitude of the cytotoxic responses
observed in vaccine recipients compared to chronically infected
patients might reflect very low precursor frequencies in the context
of vaccination. This could result in comparatively lower responses
after 6-day stimulation even if proliferative capacity remains
intact. As an initial evaluation, we correlated the magnitude of the
cytotoxic responses with the starting frequencies of CD8+T-cells
producing interferon-gamma (IFNc) and/or IL2, which had been
measured in intracellular cytokine staining (ICS) assays following
6-hour stimulation with HIV Nef, Gag or Pol peptide pools.
PBMC were obtained from similar time points for use in both the
ICS and cytotoxicity analyses. Since these responses had been
measured against pools of potential T-cell epitopes (PTE) in the
071 study and pools of 15mer peptides overlapping in sequence by
11 amino acids in the 502 study, we evaluated them separately,
even though these responses did not differ significantly between
the vaccine groups by either method (median total IFNc+and/or
IL2+CD8+T-cells 0.54% versus 0.57%, respectively, p.0.5;
median IL2+CD8+T-cells 0.08% versus 0.07%, respectively,
p.0.5; median total IFNc+and/or IL2+CD4+T-cells 0.12%
versus 0.04%, respectively, p.0.5; median total IL2+CD4+T-
cells 0.08% versus 0.0%, respectively, p=0.26; data not shown).
In the 071 study (n=19), ICE did not correlate with either the
total frequencies of IFNc+and/or IL2+CD8+T-cells (p=0.2) or
the IL2-secreting subset (p=0.25; data not shown). It also did not
correlate with the total frequencies of IFNc+and/or IL2+CD4+
T-cells (p.0.5) or IL2+CD4+T-cells (p.0.5; data not shown).
Except for a significant correlation between ICE and the
frequencies of IL2+CD4+T-cells (r=0.72, p=0.04), similar
results were observed in analyses performed among vaccine
recipients in the 502 study (n=10; data not shown). Therefore,
among those responses evaluated in both assays, the frequencies of
cytokine-producing HIV-specific CD8+T-cells induced by this
vaccine strategy were not predictive of killing capacity following
6-day stimulation with HIV-infected CD4+T-cell targets.
To determine whether the low-level cytotoxic responses for the
vaccine group relative to those of LTNP were due solely to
diminished numbers of cells following the 6-day stimulation or also
due to reduced per-cell killing capacity, we further analyzed the
cytotoxic responses in the context of the true effector-to-target
(E:T) ratios. For this analysis, ICE responses were measured at a
standard E:T ratio of 25:1 (total day 6 CD8+T-cells to total CD4+
T-cell targets) for all individuals, and additionally at an increased
E:T ratio of 50:1 (by doubling the numbers of plated CD8+T-cells)
in 20 vaccinees from whom greater numbers of PBMC were
available. These responses were then plotted against the measured
HIV-specific E:T ratios, as previously described (see Methods)
[8,19]. Curves fit to the responses of the vaccinee group at
standard and increased E:T ratios were overlaid on historical data
generated with the cells of LTNP and viremic progressors and
were analyzed by regressing ICE responses on the log of the true
E:T ratios (Figure 3). Cytotoxic responses of LTNP were
significantly greater than those of the vaccine recipients measured
with both standard (n=31) and increased (n=20) CD8+T-cell
numbers by a constant 23% (p,0.001) and 27% (p,0.001),
respectively, over the shared E:T ratios (Figure 3). In contrast, the
differences between vaccinees and progressors at both standard
(p=0.02) and increased (p=0.05) CD8+T-cell numbers varied
with the value of the E:T ratio and were not constant over their
shared range. For example, the vaccinee curve summarizing
responses measured with increased CD8+T-cell numbers was
similar to that of progressors at the median E:T ratio of 3.1:1
(p=0.17), but was significantly, yet modestly, greater at an E:T of
5:1 (8.6%, p=0.02; Figure 3). In summary, the per-cell cytotoxic
capacity of HIV-specific CD8+T-cells derived from vaccine
recipients was consistently and significantly lower than that of
LTNP across a broad range of shared E:T ratios, but was similar
to, or only modestly greater than, the per-cell cytotoxic capacity of
Figure 2. Numbers of immunizations and time from last vaccination did not correlate with the magnitude of Ad5/HIV vaccine-
induced HIV-specific CD8+ +T-cell cytotoxic responses. (A) ICE responses were compared by the Wilcoxon two-sample test between individuals
who had received 2 (n=19) versus 3 (n=12) immunizations with the Ad5/HIV vaccine through protocols 071 or 502, respectively. Horizontal lines
represent median values. (B) ICE responses did not correlate with the number of days following the last vaccination (p.0.5). Correlation was
determined by the Spearman rank method.
HIV-Specific Cytotoxic Capacity in Vaccinees
PLoS Pathogens | www.plospathogens.org5 February 2011 | Volume 7 | Issue 2 | e1002002
HIV-specific CD8+T-cell proliferation and perforin
expression correlate with cytotoxic capacity in recipients
of an Ad5/HIV vaccine
The HIV-specific CD8+T-cell cytotoxic responses of recipients
of this immunogenic Ad5/HIV vaccine could be distinguished
from the responses of HIV-negative individuals and LTNP, but
were more comparable to those of progressors. We had previously
shown in the setting of chronic infection that cytotoxicity directly
correlated with proliferative capacity and expression of the
cytotoxic proteins contained within cytotoxic granules [8,19].
Given these findings, we explored the proliferative capacity and
perforin expression of the HIV-specific CD8+T-cells of vaccinees.
CD8+T-cells were CFSE-labeled and incubated for 6 days in
parallel with non-labeled cells that were to be used as effectors in
the cytotoxicity assays. Following a 6-day incubation with
autologous HIV-infected CD4+T-cell targets, the CFSE-labeled
cells were fixed, stained for perforin and analyzed by flow
cytometry for proliferation and intracellular perforin expression
(Figure 4A–D). Consistent with prior work, CD8+
proliferation and perforin expression were strongly directly
correlated with each other (R=0.96, p,0.001; data not shown).
In analyses with cells from all patient groups, including the cells of
the recipients of the Ad5/HIV vaccine, ICE was strongly directly
correlated with both CD8+
Figure 4C, D). Focusing the analysis on the responses of vaccine
recipients, CD8+T-cell proliferation and perforin expression were
again strongly directly correlated with each other (R=0.96,
p,0.001; data not shown) and with ICE (R=0.74, p=0.001 and
R=0.81, p,0.001, respectively; Figure 4C, D). These findings
suggest that the same cascade of events that has been associated
with maximal cytotoxic capacity in chronically infected patients
also occurs in vaccine recipients. Interestingly, however, the
proliferative responses and perforin expression of the CD8+T-cells
of some of the vaccine recipients were disproportionately high for
the degree of cytotoxicity and overlapped the responses of LTNP
(Figure 4C, D). That is, their cytotoxic responses were lower than
T-cell proliferation (R=0.87,
might be predicted by HIV-specific CD8+T-cell proliferative
capacity and intracellular perforin expression, which contrasts
with observations made in chronically infected patients. These
data suggest that although proliferative capacity and perforin
expression are correlated with cytotoxic capacity, these correla-
tions are not as tight as those observed in chronic infection.
Importantly, the proliferative capacity and perforin expression of
CD8+T-cells of some vaccinees overlapped with those of LTNP
although there were considerable differences in cytotoxic capacity.
HIV-specific CD8+T-cell cytotoxic capacity in Ad5/HIV
vaccine recipients was higher among individuals with
HLA class I alleles associated with nonprogressive HIV
Given the preliminary data showing a trend towards an
association between HLA haplotype and viral load in vaccinee
cases of the Step trial (Nicole Frahm, personal communication), it
was of interest to examine the cytotoxic capacity of vaccinees in
the present study when stratified by HLA type. Interestingly, the
cytotoxic responses of vaccine recipients carrying HLA class I
alleles that have been associated with nonprogressive HIV
infection, e.g., B*27, B*57 and B*58 (n=11), were significantly
greater than those of individuals not possessing these alleles
(n=20; median ICE 48.7% versus 25.4%, respectively, p=0.001;
Figure 5). Importantly, none of the individuals bearing these
protective alleles exhibited a low response. These results suggest
that, in contrast to patients not expressing protective alleles,
vaccination in individuals carrying these alleles primes for greater
HIV-specific CD8+T-cell cytotoxic capacity.
In the present study, we examined the cytotoxic capacity of
HIV-specific CD8+T-cells, in response to primary autologous
HIV-infected CD4+T-cell targets, in samples from HIV-negative
individuals vaccinated with a replication-incompetent adenovirus
serotype 5 HIV vaccine. In prior studies, immunization with this
Figure 3. The HIV-specific CD8+ +T-cells of Ad5/HIV vaccinees exhibited per-cell cytotoxic capacity that was significantly lower than
that of LTNP but only somewhat higher than that of progressors. Cytotoxic responses mediated by day 6 CD8+T-cells were measured by ICE
for all individuals at a standard E:T ratio of 25:1 or an increased E:T ratio of 50:1 (total day 6 CD8+T-cells to total CD4+T-cell targets) and subsequently
plotted against the true E:T ratios based on measurements of IFN-c-secreting CD8+T-cells and p24-expressing target cells, respectively. Curves
represent trends for responses of LTNP (n=18, red), viremic progressors (n=19, blue), Ad5/HIV vaccinees measured at the standard E:T ratio of 25:1
(n=31, open green) and a subset of Ad5/HIV vaccinees also measured at an increased E:T ratio of 50:1 (n=20, open black). Regression analysis,
analysis of covariance and repeated measures were used to quantify the differences in ICE among LTNP, progressors and Ad5/HIV vaccinees over the
range of logged E:T ratios, at the median log E:T ratio of the combined groups and at the log E:T ratio of 5:1. ICE of LTNP differed by constant
amounts from that of vaccinees over the shared range of true E:T ratios measured with either standard (p,0.001) or increased (p,0.001) numbers of
CD8+T-cells. However, the differences between progressor and vaccinee ICE trend lines at standard (p=0.02) and increased (p=0.05) numbers of
CD8+T-cells depended on the value of the E:T ratio over the shared range. See text for more details.
HIV-Specific Cytotoxic Capacity in Vaccinees
PLoS Pathogens | www.plospathogens.org6February 2011 | Volume 7 | Issue 2 | e1002002
vaccine did not diminish the rate of HIV infection compared to
placebo recipients, nor did it lead to reductions in plasma HIV
RNA levels among newly infected persons . In our analyses,
readily detectable HIV-specific CD8+T-cell cytotoxic responses
were observed in vaccinees based on measurements of GrB target
cell activity and infected CD4+T-cell elimination. Significant
responses were present a median of 331 days following the last
immunization, confirming that long-lived memory cells had been
induced with this vaccine strategy. However, the recall cytotoxic
capacity of the HIV-specific CD8+T-cells of vaccinees was modest
and overlapped with that of progressors. The magnitude of the
cytotoxic responses was not related to the number of vaccinations,
nor did it correlate with the percentages of cytokine-secreting
T-cells determined by ICS assays. Importantly, we did observe
higher cytotoxic responses in vaccine recipients carrying HLA
class I alleles that have been associated with immune control over
HIV replication, HLA B*27, B*57 or B*58.
Given the lack of an association between traditional measures of
HIV-specific T-cell frequencies such as ELISPOT or ICS and
immunologic control of HIV after vaccination, there has been
increasing interest in measurements of other HIV-specific T-cell
functions. Significant activity was observed with viral inhibition
assays in recipients of a DNA prime/recombinant Ad5 boost
vaccine regimen [32,33]. Spentzou et al. observed relatively low
but detectable inhibition by CD8+T-cells from 7 participants that
had received a DNA-recombinant Ad5 prime-boost regimen .
Freel et al. observed low virus inhibition in 40 participants
vaccinated with a similar DNA prime/recombinant Ad5 boost
regimen . Many of these responses were below the level of
detection and the responses of vaccinees were below those of
progressors and nonprogressors. Of note, the response of chronic
progressors was similar to those of virus controllers .
Differences between these results and ours may be attributed to
differences in vaccine regimen and cohort selection criteria
[8,19,33]. In addition, they may be attributable to the assays
used. Although granule exocytosis-mediated killing may be an
important contributor in virus inhibition assays, the latter may also
measure the effects of CD8+T-cell proliferation, chemokine or
suppressor factor secretion, or cytotoxicity mediated by other
mechanisms. Nonetheless, it will be important over the coming
years to follow each of these assays for their ability to predict
vaccine-induced immunologic control of HIV and for potential
clues regarding the mechanism.
Although some HIV-specific CD8+T-cell cytotoxic capacity
was induced by the vaccine approach in the current study, the
magnitude necessary for immunologic restriction of HIV remains
unclear. Since participants were not infected after vaccination, it
was reasonable to suspect their precursor frequencies were most
Figure 4. HIV-specific CD8+ +T-cell proliferation and perforin expression correlated with cytotoxic capacity in recipients of an Ad5/
HIV vaccine. (A, B) Flow cytometry plots of a representative LTNP (top row) and Ad5/HIV vaccine recipient (bottom row) depicting CD8+T-cell
proliferation (A) and CD8+T-cell perforin expression (B) following 6-day stimulation of CFSE-labeled PBMC with uninfected (left columns) or HIVSF162-
infected (right columns) autologous CD4+T-cell targets. Plots are gated on CD8+T-cells. Net HIV-specific CD8+T-cell proliferation (sum of upper left
and lower left quadrants in A, right column) and net CD8+T-cell perforin expression (as determined by subtracting background perforin expression
measured in cells that had been stimulated with uninfected targets; B, left column) are shown in red font. (C, D) Summary data of CD8+T-cell
proliferation (C) and perforin expression (D) are shown for LTNP (red symbols, n=7), progressors (blue symbols, n=10), HIV-negative individuals
(black symbols, n=10) and Ad5/HIV vaccinees (green symbols, n=19), including 5 individuals carrying the protective HLA class I alleles B*27, B*57 or
B*58 (solid green symbols). Data are representative of at least two experiments. Statistical analyses were performed using the Spearman correlation.
HIV-Specific Cytotoxic Capacity in Vaccinees
PLoS Pathogens | www.plospathogens.org7 February 2011 | Volume 7 | Issue 2 | e1002002
likely lower than those of chronically infected patients. Although
this vaccine approach induced pre-challenge levels of Mamu A01-
p11CM MHC tetramer+CD8+T-cells in the peripheral blood of
rhesus macaques that reached 2% in the Ad5/SIV only arm and
levels ranging from 5–25% when preceded by a DNA/CRL1005
prime, the Ad5/HIV vaccine induced only a median response of
0.4–1% HIV-specific CD8+T-cells based upon ICS in humans in
a prior study and 0.5–0.6% in the present study [25,27]. However,
it is clear that the modest cytotoxic capacity observed in vaccinees
was not simply due to low frequencies of HIV-specific CD8+
T-cells. The frequencies of HIV-specific CD8+T-cells did not
correlate with cytotoxic capacity. Furthermore, some of the
individuals with the lowest frequencies of HIV-specific T-cells
exhibited the highest CD8+T-cell cytotoxic responses several years
Another potential factor contributing to the diminished HIV-
specific CD8+T-cell-mediated cytotoxicity observed in vaccinees
relative to LTNP might relate to response breadth. That is, there
may be additional CD8+T-cell responses in LTNP targeting
proteins outside of the 3 genes included in the vaccine, which
might have led to more efficient elimination of HIV-infected
CD4+T-cell targets. This seems unlikely to be the case, however,
since the bulk of the cytokine secretory and proliferative responses
measured in LTNP have been primarily directed against highly
conserved epitopes contained within Nef, Gag and Pol, with only
minimal contribution made by responses targeting other gene
In order to investigate the cytotoxic responses of the various
groups in greater detail, we analyzed them in the context of the
true measured E:T ratios following 6-day re-stimulation and found
that the per-cell killing capacity for the group of vaccine recipients
again remained only slightly higher than that of progressors, but
significantly lower than that of LTNP. Although it is not
necessarily expected that the CD8+T-cells of vaccinees would
achieve the cytotoxic capacity of chronically infected LTNP, the
results of the present study suggest that the human immune
response is capable of higher responses. The observation that
vaccinee per-cell cytotoxic capacity increased at higher E:T ratios
and became more divergent from that of progressors also suggests
that induction of higher cytotoxic responses might be attainable
with improved vaccine design strategies. These results suggest a
threshold level of cytotoxic capacity might have been achieved
only in a small subset of patients with protective alleles in this
vaccination scheme. Further investigation of the cytotoxic
responses directed against other viral infections that have been
cleared or are controlled by the host may provide a better context
in which to interpret the responses measured in this study. Most
importantly, further evaluation of vaccinee cases may reveal
whether these measurements of HIV-specific CD8+
cytotoxic capacity can accurately predict immune control over
HIV following vaccination.
In the present study, we did observe a clear effect of protective
HLA alleles in priming HIV-specific CD8+T-cell cytotoxic
capacity. However, whether these alleles, in the context of
vaccination, are associated with qualitatively different CD8+
T-cell responses or immunologic control has been examined in
some prior work with mixed results [28,33]. Preliminary data from
the Step study suggested that, after HIV infection, vaccinees with
the protective HLA alleles B*57, B*58 and B*27 may have lower
HIV RNA levels compared to those with protective alleles that
received placebo (Nicole Frahm, personal communication).
However, in a subsequent analysis, although those with protective
alleles had lower viral loads overall, the difference between
placebo and vaccinee cases with protective alleles did not achieve
statistical significance (Nicole Frahm, personal communication).
Kaslow et al observed that CD8+T-cells restricted by protective
alleles dominate the response to vaccination with an ALVAC-HIV
recombinant canarypox . In addition, Freel et al. observed
some increased HIV inhibition in cells from vaccinees with B*27
or B*57 compared to those who lacked these alleles. However, this
was only true of NL4-3 and WEAU viruses . Taken together,
these data suggest that protective alleles may function to more
efficiently prime HIV-specific CD8+T-cell cytotoxic capacity. The
precise mechanism underlying this association, however, is
currently unclear. CD8+T-cell responses restricted by HLA B27
and 57 predominate during acute infection  and are more
likely to maintain the capacity to proliferate after prolonged
infection than responses restricted by other alleles [34,36].
Enhanced induction of CD8+T-cell proliferative responses leading
downstream to increased cytotoxic capacity might result from a
greater ability of infected cells to stimulate naı ¨ve CD8+T-cells.
Alternatively, it might result from an enhanced ability of CD8+T-
cells to respond to infected cells due to interactions related to killer
immunoglobulin-like receptor (KIR) ligation, co-stimulatory signal
requirements, or a diverse naı ¨ve T-cell repertoire. In any event,
whether the increased cytotoxic capacity observed in the present
study translates into better immunologic control will need to be
monitored in future trials.
Although the HIV-specific CD8+T-cell cytotoxic capacity of
vaccinees was less than that observed among LTNP, the cells of
some vaccinees rapidly expanded and produced perforin in
response to HIV-infected T-cells. In chronic infection, we have
observed a very tight correlation between proliferative capacity
and perforin expression as we did in the present study .
However, a range of proliferative responses and perforin
expression was observed in vaccinees that, in some cases,
overlapped those of LTNP even though these cells did not exhibit
cytotoxic responses of similar magnitudes as those of LTNP. It is
unlikely that such a large fraction of vaccinees would exhibit
Figure 5. HIV-specific CD8+ +T-cell cytotoxic capacity in Ad5/HIV
vaccine recipients was higher among individuals with protec-
tive HLA class I alleles. ICE responses mediated by day 6 CD8+T-cells
of vaccine recipients who possess the HLA class I alleles B*27, B*57 or
B*58 that have been shown to be associated with nonprogressive HIV
infection (n=11) versus those of vaccine recipients not possessing
these protective alleles (n=20) was compared by the Wilcoxon two-
sample test. Horizontal lines represent median values. Data are
representative of at least two experiments.
HIV-Specific Cytotoxic Capacity in Vaccinees
PLoS Pathogens | www.plospathogens.org8 February 2011 | Volume 7 | Issue 2 | e1002002
immunologic control upon infection. Thus, proliferation or
perforin expression, at least under these experimental conditions,
are unlikely to be better predictors of immunologic control than
cytotoxic capacity in the context of vaccination. The observation
of cells with proliferative potential and the ability to make perforin
similar to those of LTNP but with only modest cytotoxic capacity
may relate to vaccine vector, dose, vaccine administration
schedule and timing of post-vaccination PBMC collection for use
in the immunologic assays. These might have an impact on the
pattern of differentiation or maturation of HIV-specific CD8+T-
cells, which could influence direct measures of killing capacity.
Additional studies are currently under way to further characterize
the functional capabilities of these cells.
Although many challenges lie ahead, it appears possible that T-
cell based immunogens may provide some activity to reduce viral
load upon infection. Currently, antibody-based vaccine approach-
es are receiving increased emphasis with the reported reduction in
HIV acquisition observed in the RV144 trial . However, an
effective T-cell response would potentially complement an
effective humoral immune response by reducing viral load in
vaccinees that become infected . It may also have the effect of
reducing the transmission of antibody resistant mutants. Immu-
nologic control in chronically infected LTNP is durable and has
lasted more than 25 years in many cases (reviewed in ). In
addition, many patients lack known protective MHC alleles
suggesting these are not an absolute requirement for durable
immunologic control . Evidence of durable immunologic
control that extends beyond known protective alleles has also
been provided by recent studies in the SIV infection model.
Cellular immune responses elicited by infection with recombinant
rhesus cytomegalovirus encoding SIV antigens can provide
resistance to SIV infection on repeated limiting-dose intrarectal
challenge . Ongoing studies are examining whether cytotoxic
capacity is an important mechanism of immune control in the
rhesus macaque-SIV infection model (Daniel Mendoza, unpub-
lished observations). Importantly, our assay will be applied to
participants in the Step study who subsequently acquired HIV
infection post-vaccination, including a group of individuals who
are restricting HIV replication to the limits of detection in
currently available viral load assays. These types of analyses will
begin to determine the utility of measurements of cytotoxic
capacity in predicting vaccine efficacy, and provide further insight
into the mechanisms of immunologic control of HIV.
HIV-specific CD8+T-cell cytotoxic responses were
measured by granzyme B target cell activity and infected CD4+T-
cell elimination in chronically infected patients and Ad5/HIV
vaccine recipients. (A) Following incubation with day 6 autologous
CD8+T-cells, granzyme (Gr) B activity in uninfected (left column)
or HIV-infected CD4+T-cell lymphoblast targets (right column) is
shown in a representative LTNP (top row) and an Ad5/HIV
vaccine recipient (bottom row). Plots are gated on live targets
based on staining with a LIVE/DEAD Fixable Violet Stain (see
Methods). Net GrB target cell activity after subtracting back-
ground values (i.e., responses in uninfected targets, left column) is
shown in red font. (B) Cells from A after fixation, permeabilization
and staining for CD4 and intracellular p24 expression. Quadrants
indicate percentages of gated targets. Infected CD4 elimination
(ICE) is shown in red font, which was calculated with p24+targets
(sum of upper quadrants) as described in the Methods.
Found at: doi:10.1371/journal.ppat.1002002.s001 (TIFF)
Found at: doi:10.1371/journal.ppat.1002002.s002 (DOCX)
Ad5/HIV vaccine recipient characteristics.
Found at: doi:10.1371/journal.ppat.1002002.s003 (DOCX)
Characteristics of HIV-infected patients.
Conceived and designed the experiments: SAM MC. Performed the
experiments: JER AMB TG DM AP. Analyzed the data: CWH JER AMB
TG SAM. Contributed reagents/materials/analysis tools: NAC AP NF AD
MJM. Wrote the paper: SAM MC.
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HIV-Specific Cytotoxic Capacity in Vaccinees
PLoS Pathogens | www.plospathogens.org10 February 2011 | Volume 7 | Issue 2 | e1002002