Strong human endogenous retrovirus-specific T cell responses are associated with control of HIV-1 in chronic infection.
ABSTRACT Eight percent of the human genome is composed of human endogenous retroviruses (HERVs), which are thought to be inactive remnants of ancient infections. Previously, we showed that individuals with early HIV-1 infection have stronger anti-HERV T cell responses than uninfected controls. In this study, we investigated whether these responses persist in chronic HIV-1 infection and whether they have a role in the control of HIV-1. Peripheral blood mononuclear cells (PBMCs) from 88 subjects diagnosed with HIV-1 infection for at least 1 year (median duration of diagnosis, 13 years) were tested for responses against HERV peptides in gamma interferon (IFN-γ) enzyme immunospot (ELISPOT) assays. Individuals who control HIV-1 viremia without highly active antiretroviral therapy (HAART) had stronger and broader HERV-specific T cell responses than HAART-suppressed patients, virologic noncontrollers, immunologic progressors, and uninfected controls (P < 0.05 for each pairwise comparison). In addition, the magnitude of the anti-HERV T cell response was inversely correlated with HIV-1 viral load (r(2) = 0.197, P = 0.0002) and associated with higher CD4(+) T cell counts (r(2) = 0.072, P = 0.027) in untreated patients. Flow cytometric analyses of an HLA-B51-restricted CD8(+) HERV response in one HIV-1-infected individual revealed a less activated and more differentiated phenotype than that stimulated by a homologous HIV-1 peptide. HLA-B51 tetramer dual staining within this individual confirmed two different T cell populations corresponding to these HERV and HIV-1 epitopes, ruling out cross-reactivity. These findings suggest a possible role for anti-HERV immunity in the control of chronic HIV-1 infection and provide support for a larger effort to design an HIV-1 vaccine that targets conserved antigens such as HERV.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: Human endogenous retroviruses (HERVs) are remnants of ancestral infections and chromosomally integrated in all cells of an individual, are transmitted only vertically and are defective in viral replication. However enhanced expression of HERV-K accompanied by the emergence of anti-HERV-K-directed immune responses has been observed inter-alia in HIV-infected individuals and tumor patients. Therefore HERV-K might serve as a tumor-specific antigen or even as a constant target for the development of an HIV vaccine. To verify our hypothesis, we tested the immunogenicity of HERV-K Gag by using a recombinant vaccinia virus (MVA-HKcon) expressing the HERV-K Gag protein and established an animal model to test its vaccination efficacy. Murine renal carcinoma cells (Renca) were genetically altered to express E. coli beta-galactosidase (RLZ cells) and the HERV-K Gag protein (RLZ-HKGag cells). Subcutaneous application of RLZ-HKGag cells into syngenic BALB/c mice resulted in the formation of local tumors in MVA vaccinated mice. MVA-HKcon vaccination reduced the tumor growth. Furthermore, intravenous injection of RLZ-HKGag cells led to the formation of pulmonary metastases. Vaccination of tumor-bearing mice with MVA-HKcon drastically reduced the number of pulmonary RLZ-HKGag tumor nodules compared to vaccination with wild-type MVA. The data demonstrate that HERV-K Gag is a useful target for vaccine development and might offer new treatment opportunities for cancer patients. .Virology Journal 03/2014; 11(1):58. · 2.09 Impact Factor
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ABSTRACT: Human Endogenous Retroviruses (HERVs) comprise about 8% of the human genome and have lost their ability to replicate or to produce infectious particles after having accumulated mutations over time. We assessed the kinetics of expression of HERV-K (HML-2) Envelope mRNA transcript and surface unit (SU) and transmembrane (TM) subunit proteins during HIV-1 infection. We also mapped the specificity of the humoral response to HERV-K (HML-2) Envelope protein in HIV-1 infected subjects at different stages of disease, and correlated the response with plasma viral load. We found that HIV-1 modified HERV-K (HML-2) Env mRNA expression, resulting in the expression of a fully N-glycosylated HERV-K (HML-2) envelope protein on the cell surface. Serological mapping of HERV-K (HML-2) envelope protein linear epitopes revealed two major immunogenic domains, one on SU and another on the ectodomain of TM. The titers of HERV-K (HML-2) TM antibodies were dramatically increased in HIV-1 infected subjects (p < 0.0001). HIV-1 infected adults who control HIV-1 in the absence of therapy ("elite" controllers) had a higher titer response against TM compared to antiretroviral-treated adults (p < 0.0001) and uninfected adults (p < 0.0001). These data collectively suggest that HIV-1 infection induces fully glycosylated HERV-K (HML-2) envelope TM protein to which antibodies are induced. These anti-HERV-K (HML-2) TM antibodies are a potential marker of HIV-1 infection, and are at higher titer in elite controllers. HERV-K (HML-2) envelope TM protein may be a new therapeutic target in HIV-1 infection.Retrovirology 01/2014; 11(1):10. · 5.66 Impact Factor
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ABSTRACT: The enormous sequence diversity of HIV remains a major roadblock to the development of a prophylactic vaccine and new approaches to induce protective immunity are needed. Endogenous retrotransposable elements (ERE) such as endogenous retrovirus K (ERV)-K and long interspersed nuclear element-1 (LINE-1) are activated during HIV-1-infection and could represent stable, surrogate targets to eliminate HIV-1-infected cells. Here, we explored the hypothesis that vaccination against ERE would protect macaques from acquisition and replication of simian immunodeficiency virus (SIV). Following vaccination with antigens derived from LINE-1 and ERV-K consensus sequences, animals mounted immune responses that failed to delay acquisition of SIVsmE660. We observed no differences in acute or set point viral loads between ERE-vaccinated and control animals suggesting that ERE-specific responses were not protective. Indeed, ERE-specific T cells failed to expand anamnestically in vivo following infection with SIVsmE660 and did not recognize SIV-infected targets in vitro, in agreement with no significant induction of targeted ERE mRNA by SIV in macaque CD4+ T cells. Instead, lower infection rates and viral loads correlated significantly to protective TRIM5α alleles. Cumulatively, these data demonstrate that vaccination against the selected ERE consensus sequences in macaques did not lead to immune-mediated recognition and killing of SIV-infected cells, as has been shown for HIV-infected human cells using patient-derived HERV-K-specific T cells. Thus, further research is required to identify the specific nonhuman primate EREs and retroviruses that recapitulate the activity of HIV-1 in human cells. These results also highlight the complexity in translating observations of the interplay between HIV-1 and human EREs to animal models.PLoS ONE 01/2014; 9(3):e92012. · 3.73 Impact Factor
Published Ahead of Print 27 April 2011.
2011, 85(14):6977. DOI: 10.1128/JVI.00179-11.
Steven G. Deeks and Douglas F. Nixon
Reyes-Terán, Mario A. Ostrowski, Frederick M. Hecht,
Jeffrey N. Martin, Vanessa A. York, Gerald Spotts, Gustavo
Ormsby, Liyen Loh, R. Brad Jones, Keith E. Garrison,
Lishomwa C. Ndhlovu, Rachel Lown-Hecht, Christopher E.
Devi SenGupta, Ravi Tandon, Raphaella G. S. Vieira,
Associated with Control of HIV-1 in Chronic
Retrovirus-Specific T Cell Responses Are
Strong Human Endogenous
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JOURNAL OF VIROLOGY, July 2011, p. 6977–6985
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 14
Strong Human Endogenous Retrovirus-Specific T Cell Responses Are
Associated with Control of HIV-1 in Chronic Infection?†‡
Devi SenGupta,1* Ravi Tandon,1Raphaella G. S. Vieira,1Lishomwa C. Ndhlovu,1Rachel Lown-Hecht,1
Christopher E. Ormsby,2Liyen Loh,1R. Brad Jones,3Keith E. Garrison,1Jeffrey N. Martin,4
Vanessa A. York,1Gerald Spotts,5Gustavo Reyes-Tera ´n,2Mario A. Ostrowski,3
Frederick M. Hecht,5Steven G. Deeks,5and Douglas F. Nixon1
Division of Experimental Medicine, Department of Medicine, University of California, San Francisco, San Francisco, California 941101;
Center for Research in Infectious Diseases, National Institute of Respiratory Diseases, Mexico City, Federal District, Mexico CP 140802;
Department of Immunology, University of Toronto, Toronto, Ontario, Canada M5S 1A83; Department of Epidemiology and
Biostatistics, University of California, San Francisco, San Francisco, California, 941104; and Positive Health Program,
San Francisco General Hospital, San Francisco, California, 941105
Received 26 January 2011/Accepted 19 April 2011
Eight percent of the human genome is composed of human endogenous retroviruses (HERVs), which are
thought to be inactive remnants of ancient infections. Previously, we showed that individuals with early HIV-1
infection have stronger anti-HERV T cell responses than uninfected controls. In this study, we investigated
whether these responses persist in chronic HIV-1 infection and whether they have a role in the control of HIV-1.
Peripheral blood mononuclear cells (PBMCs) from 88 subjects diagnosed with HIV-1 infection for at least 1
year (median duration of diagnosis, 13 years) were tested for responses against HERV peptides in gamma
interferon (IFN-?) enzyme immunospot (ELISPOT) assays. Individuals who control HIV-1 viremia without
highly active antiretroviral therapy (HAART) had stronger and broader HERV-specific T cell responses than
HAART-suppressed patients, virologic noncontrollers, immunologic progressors, and uninfected controls (P <
0.05 for each pairwise comparison). In addition, the magnitude of the anti-HERV T cell response was inversely
correlated with HIV-1 viral load (r2? 0.197, P ? 0.0002) and associated with higher CD4?T cell counts (r2?
0.072, P ? 0.027) in untreated patients. Flow cytometric analyses of an HLA-B51-restricted CD8?HERV
response in one HIV-1-infected individual revealed a less activated and more differentiated phenotype than
that stimulated by a homologous HIV-1 peptide. HLA-B51 tetramer dual staining within this individual
confirmed two different T cell populations corresponding to these HERV and HIV-1 epitopes, ruling out
cross-reactivity. These findings suggest a possible role for anti-HERV immunity in the control of chronic HIV-1
infection and provide support for a larger effort to design an HIV-1 vaccine that targets conserved antigens
such as HERV.
Much of the recent effort in identifying immune correlates of
protection in HIV-1 has focused on defining the qualities of a
successful T cell response in individuals who maintain low or
undetectable viral loads in the absence of treatment, i.e., con-
trollers (1, 6, 9, 27). As a result of these investigations, there
have been major advances in the understanding of the immu-
nobiology of HIV-1 infection (30). However, the quest for a
fully effective vaccine is still ongoing, and the designs of many
vaccine candidates have only varied from each other slightly
(31). We have studied an area outside conventional HIV-1
vaccine strategies and explored the potential for a human en-
dogenous retrovirus (HERV)-based HIV-1 vaccine.
Transposable elements make up 45% of the human genome
(19). Human endogenous retroelements (which rely on an
RNA intermediate before integration into the genome) can be
classified into the non-long terminal repeat (LTR) class and
the LTR class, represented by endogenous retroviruses
(ERV). Many ERV entered the primate germ line as infec-
tious retroviruses at several time points during human evolu-
tion (4). Out of the six HERV superfamilies, HERV-K
(HML-2) is considered to be the youngest and most transcrip-
tionally active (18). A report in 2008 described the visualiza-
tion of HERV-K-like viral particles in the plasma of lymphoma
patients (8). A clinical study demonstrated the potential utili-
zation of another HERV family in a novel cancer immuno-
therapy product (33). In a group of patients with metastatic
renal cell carcinoma (RCC) who experienced tumor regression
after allogeneic hematopoietic stem cell transplant, RCC-re-
active donor-derived CD8?T cells directed against a 10-mer
HERV-E peptide were identified. This antigen was expressed
in RCC cells but not healthy tissues, suggesting that it could be
targeted by a new tumor peptide vaccine or by adoptively
infused peptide-specific cytotoxic T lymphocytes (CTLs) (33).
Host cells have developed mechanisms to prevent lentiviral
replication, as well as to restrict the movement of retroele-
ments in order to maintain genomic stability. Esnault et al. (11)
and others (24) reported that the host protein APOBEC3
* Corresponding author. Mailing address: UCSF Division of Exper-
imental Medicine, Building 3, Room 603, San Francisco General Hos-
pital, 1001 Potrero Avenue, San Francisco, CA 94110. Phone: (415)
206-4981. Fax: (415) 206-8091. E-mail: Devi.SenGupta@ucsf.edu.
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 27 April 2011.
‡ The authors have paid a fee to allow immediate free access to this
on March 29, 2012 by guest
restricts endogenous retroelements. The HIV-1 Vif protein
has been shown to disable APOBEC3G (13, 32), which could
lead to the activation of HERV. In support of this hypothesis,
our group and others have demonstrated that HERV-K tran-
scripts can be detected in the plasma of HIV-1-infected indi-
viduals, suggesting that HIV-1 infection can alter the biology of
HERV-K by enhancing its gene expression (7, 12).
We also previously reported that adults with early HIV-1
infection have detectable ex vivo CD8?T cell responses to
HERV antigens and that these responses are absent in healthy
subjects (12). The magnitude of the anti-HERV response is
inversely correlated with HIV-1 plasma viral load, suggesting a
potential role for these responses in the control of HIV-1 (12).
The current study examined the immunological consequences
of HERV antigen production and presentation in individuals
with chronic HIV-1. We hypothesized that subjects who are
able to control HIV-1 in the absence of highly active antiret-
roviral therapy (HAART) have stronger and more frequent
HERV-specific T cell responses than those who are unable to
control HIV-1 without HAART. We also hypothesized that
responses would be higher in individuals controlling the virus
as an apparent consequence of the host response (controllers)
than in individuals suppressing the virus as a consequence of
antiretroviral treatment. In order to elucidate a possible mech-
anism of viral control in these individuals, we compared the
functionality and phenotype of HERV-, HIV-1-, and cytomeg-
alovirus (CMV)-specific responses. The work outlined in this
study has the potential to lead to a vaccination strategy utiliz-
ing HERV-specific T cells with the capacity to kill HIV-1-
infected cells expressing HERV epitopes. It is envisioned that
such a novel vaccine would be part of a combination vaccina-
tion approach that includes stimulation of immunity to HIV-1
antigens as well as to HERV.
MATERIALS AND METHODS
Study populations. Samples of peripheral blood mononuclear cells (PBMCs)
were selected from participants in two different San Francisco-based HIV-1-
infected cohorts, Options (15) and SCOPE (17), as well as from an HIV-1-
infected cohort at the University of Toronto. Samples from HIV-1-negative
controls were obtained from 22 individuals who donated blood to the Stanford
blood bank. The study was approved by the local institutional review boards
(University of California, San Francisco [UCSF] Committee on Human Re-
search and University of Toronto Institutional Review Board), and individuals
gave written informed consent. Studies were performed on cryopreserved
PBMC samples were obtained from the following categories of HIV-1-infected
individuals: 30 untreated virologic controllers (which included 10 viremic con-
trollers with HIV-1 viral loads between 50 and 2,000 copies/ml and 20 elite
controllers with viral loads of ?50 to 75 HIV-1 copies/ml), 20 highly active
antiretroviral therapy (HAART)-suppressed patients (?50 to 75 HIV-1 copies/
ml), and 18 untreated virologic noncontrollers (?2,000 copies/ml). All had a
CD4?T cell count of ?250 cells/mm3. A fourth group of untreated HIV-1-
infected patients was also tested. These 20 individuals were defined as immuno-
logic progressors, with an HIV-1 viral load of ?2,000 copies/ml and CD4?T cell
count of ?250 cells/mm3. All 88 patients had been diagnosed with HIV-1 at least
1 year prior to inclusion in this study. There was no significant difference in the
median ages of the five groups (including HIV-1-infected and uninfected sub-
jects) or duration of diagnosis among the HIV-1-infected groups (P ? 0.05 for all
pairwise comparisons). Table 1 describes baseline subject characteristics.
PBMC preparation and storage. PBMCs were isolated by standard Ficoll-
Hypaque density gradient centrifugation on fresh blood samples and immedi-
ately cryopreserved in fetal calf serum (HyClone, Logan, UT) containing 10%
dimethyl sulfoxide (Sigma Aldrich, St. Louis, MO) in liquid nitrogen. The cryo-
preserved cells were stored in liquid nitrogen until they were used.
Peptides and tetramers. A commercially manufactured set of 28 9- to 13-mer
individual HERV peptides with ?95% purity (Genscript, Piscataway, NJ) was
used to screen individuals for anti-HERV CD8?T cell responses (see Table S1
in the supplemental material). The HERV peptides were designed using HLA
binding prediction software (with a predicted combined binding score of ?0.75
according to NetCTL, version 1.2) (20, 29). They included peptides from several
HERV families, including HERV-K, HERV-H, and HERV-L. A subset of these
peptides was used in our previous publication (12) and is marked with an asterisk
in Table S1. A set of overlapping 15-mer HERV-K Gag and Env peptides (JPT
Peptide Technologies, Berlin, Germany) was also used to comprehensively map
one elite controller’s (subject P1’s) responses. HIV-1 peptides selected from the
Gag, Nef, Env, and Pol proteins (Sigma Aldrich and Invitrogen, Carlsbad, CA)
(see Table S2) and a CMV pp65 pool of overlapping 15-mer peptides (JPT
Peptide Technologies) were also tested. All HERV peptides were used at a
concentration of 100 ?g/ml for the initial enzyme-linked immunospot
(ELISPOT) screening assays and then titrated down to final concentrations of 1
?g/ml to 10 ?g/ml for subsequent ELISPOT assays and flow cytometry experi-
ments. HLA-B51 tetramers were synthesized and provided by the NIH Tetramer
Core Facility (Atlanta, GA).
ELISPOT assays. The ELISPOT assay has been described previously (22). In
brief, 96-well plates (Millipore, Billerica, MA) were coated with human mono-
clonal anti-interferon gamma (IFN-?) immunoglobulin (Mabtech, Mariemont,
OH). After plates were washed and blocked with 10% fetal calf serum, PBMCs
were added at a concentration of 105cells per well. Duplicate wells were pre-
pared for each experimental condition. Spot totals for duplicate wells were
averaged, and all spot numbers were normalized to numbers of IFN-? spot-
forming units (SFU) per million PBMCs (SPM). The spot values from medium
control wells were subtracted, after which a positive response to a peptide was
defined as ?50 SPM and ?2 times the medium control value. The total magni-
TABLE 1. Characteristics of study subjects
Participant category (n)
Ethnicity and no.
Median CD4?T cell
Median HIV-1 viral load
Controller (30)46.5 (40.5–52) 237 C, 13; L, 4; A, 1;
AA, 10; NA, 2
C, 11; L, 5; A, 1;
C, 9; A, 1; AA, 8
13 (6–18) 794 (569–1,059) 50 (40–302)
HAART-suppressed (20)48 (39.5–51)173 13 (9.8–17)686 (568–1,069) 75 (50–75)
Virologic noncontroller (18)49.5 (38.5–54)153 14 (8.5–20)523 (370.5–744.5) 37,568 (17,573–69,763)
Immunologic progressors (20) 43 (37–47)173C, 10; L, 5; A, 1;
AA, 3; NA, 1
C, 8; L, 2; A, 3
8 (5–16) 200 (144.5–231.5)150,974 (89,532–230,970)
HIV-1 negative (22)c
aF, female; M, male.
bC, Caucasian; L, Latino/Hispanic; A, Asian; AA, African-American; NA, Native American.
cSome information about the gender and ethnicity of the HIV-1-negative group was not available.
6978SENGUPTA ET AL.J. VIROL.
on March 29, 2012 by guest
tude of the HERV T cell response was calculated by adding up all of the
individual peptide SPM values.
HLA restriction. The HLA restriction of one of the most immunogenic HERV
peptides was determined using patient-derived PBMCs incubated with HERV
peptide-pulsed and unpulsed single-HLA allele transfectant B cells as targets
(provided by Lewis Lanier, University of California, San Francisco, CA) in an
ELISPOT assay. B cell transfectants at a concentration of 104cells per well were
pulsed with peptide for 1 h at 37°C and then washed three times before being
added into a standard ELISPOT assay with PBMCs. Control conditions included
PBMCs incubated with HLA-mismatched (B51?) targets with or without peptide
pulsing and HLA-matched (B51?) targets with or without peptide pulsing. As an
additional control, B cell transfectants with or without peptide pulsing were also
tested in the ELISPOT assay without PBMCs.
Cell surface and intracellular staining by flow cytometry. PBMCs from an
HIV-1-infected individual (subject P2) were stimulated with the peptides
HERV-K FAFTIPAI ([FI8] from the reverse transcriptase region), HIV-1 TA
FTIPSI (TI8), or a CMV pp65 peptide pool for 6 h at 37°C in the presence of
anti-CD49d (BD Biosciences, San Jose, CA), Golgi Stop (BD Biosciences), and
brefeldin A (Sigma Aldrich). Control conditions included unstimulated cells
(negative control) and cells stimulated with anti-CD3 antibody (positive control).
After incubation, cells were washed with fluorescence-activated cell sorting
(FACS) buffer (phosphate-buffered saline with 1% bovine serum albumin; Sigma
Aldrich). To determine phenotype and function of antigen-specific cells, 106
antigen-stimulated PBMCs were surface stained with fluorophore-conjugated
antibodies to anti-CD3, -CD4, -CD8, -CD27, and -CD28 (all BD Biosciences).
Following cell surface staining, PBMCs were washed, fixed with 1% paraformal-
dehyde (Polyscience, Niles, IL), and permeabilized with Perm Buffer (BD Bio-
sciences). They were then stained with antibodies against intracellular cytokines
IFN-? and tumor necrosis factor alpha (TNF-?). A second phenotyping (T cell
activation) panel consisted of cell surface markers for CD3, CD4, CD8, CD38,
and intracellular cytokines IFN-? and TNF-?. An amine aqua dye (Invitrogen)
was also included to discriminate between live and dead cells. Following staining,
PBMCs were washed with FACS buffer, fixed in paraformaldehyde, and stored at
4°C until analysis.
An HLA-B51-HERV-K FAFTIPAI-allophycocyanin (APC) tetramer and a
B51-HIV-1 TAFTIPSI-phycoerythrin (PE) tetramer (NIH Tetramer Core Facil-
ity, Atlanta, GA) were used to stain PBMCs from an HIV-1-infected subject (P2)
and a healthy HIV-1-negative control. Each tetramer was used at a dilution of
1:1,000 (based on prior determination of the optimal titration). The tetramer
dual staining was carried out at 37°C in the dark for 30 min. The cells were then
washed and stained with fluorophore-conjugated anti-CD3, -CD8, -CD14, and
-CD19 antibodies (the latter two to exclude monocytes and B cells) and an amine
aqua dye to exclude dead cells.
For all flow cytometry experiments, data were acquired with an LSR-II system
(Becton Dickinson). At least 100,000 events were collected and analyzed with
FlowJo software, version 9.0 (Tree Star, Ashland, OR).
Statistical analysis. Subject characteristics (age, duration of diagnosis, HIV-1
viral load, and CD4?T cell count), the total magnitudes of ELISPOT assay
responses (in SPM), and the total number of positive responses were compared
between groups using the Kruskal-Wallis, Dunn’s multiple comparison, and
two-sided Mann-Whitney U tests. Linear regression and Spearman correlation
analyses were used to measure associations between HIV-1 viral load and mag-
nitude and breadth of the HERV response, HIV-1 viral load and magnitude of
the HIV-1 response, the magnitudes of the HIV-1 response and the HERV
response, and the magnitude of the HERV response and CD4?T cell count in
HIV-1-infected individuals not on treatment. All tests were conducted using
GraphPad Prism, version 4.00 (GraphPad Software, San Diego, CA), with the
statistical significance of the findings set at a P value of less than 0.05.
HIV-1 controllers have stronger HERV-specific responses
than other chronically infected subjects. We measured T cell
responses against HERV using a set of manufactured peptides
selected from three HERV families (HERV-K, HERV-L, and
HERV-H). The total magnitude of the HERV-specific T cell
response was measured as the sum of all of the single peptide
responses for each individual. The median total magnitude of
HERV-specific T cell responses among HIV-1-negative sub-
jects was 122.1 SPM (interquartile range [IQR], 62.5 to 165
SPM). Individuals controlling HIV-1 viremia in the absence of
treatment (controllers) had a median total response magnitude
of 571.4 SPM (IQR, 240 to 1,395 SPM). This was significantly
higher than the total responses of the HIV-1-negative subjects
(P ? 0.0001), the HAART-suppressed group (median, 270.3
SPM; IQR, 87.6 to 422.5 SPM; P ? 0.05), the noncontroller
group (median, 181.3 SPM; IQR, 32.5 to 327.5 SPM; P ? 0.05),
and the progressors (median, 77.5 SPM; IQR, 22.5 to 175; P ?
0.0001) (Fig. 1A). HIV-1-infected controllers also had a sig-
nificantly higher number (breadth) of HERV-specific re-
FIG. 1. (A) The total HERV response magnitude of HIV-1 con-
trollers was significantly higher than that of HAART-suppressed pa-
tients (*, P ? 0.05), noncontrollers (*, P ? 0.05), progressors (***,
P ? 0.0001), and HIV-1-negative subjects (***, P ? 0.0001), as mea-
sured in IFN-? ELISPOT assays. SFU, spot-forming units; SPM, spot-
forming units per million PBMCs. (B) HIV-1 controllers had a signif-
icantly higher number of positive HERV-specific T cell responses than
HAART-suppressed patients (*, P ? 0.05), noncontrollers (**, P ?
0.01), progressors (***, P ? 0.0001), and HIV-negative subjects (***,
P ? 0.0001). A positive response was defined as ?50 SFU/million
PBMC and ?2 times the negative-control value (after subtraction of
the value of the negative control). P values are shown only for statis-
tically significant pairwise comparisons (derived from the Mann-Whit-
ney test and the Dunn’s multiple comparison test as posttests to the
Kruskal-Wallis test). Medians with interquartile range are shown for
VOL. 85, 2011 HERV-SPECIFIC T CELL RESPONSES IN HIV-1 INFECTION6979
on March 29, 2012 by guest
sponses than the HIV-1-negative individuals (P ? 0.0001), the
HIV-1-infected noncontrollers (P ? 0.01), progressors (P ?
Untreated HIV-1-infected subjects with preserved CD4?T
cell counts do not differ in the magnitude of their HIV-1 and
CMV responses. In order to characterize the baseline immune
function of the HIV-1-infected subjects, responses to an HIV-1
peptide pool (which included Gag, Nef, Env, and Pol se-
quences) and a CMV pp65 peptide pool were assessed in a
subset of individuals for whom adequate PBMC samples were
available. There was no significant difference in the CMV pp65
pool response magnitude among the three HIV-1-infected
groups with preserved CD4?T cell counts (P ? 0.05 for all
pairwise comparisons) (see Fig. S1A in the supplemental ma-
terial). However, the progressors had a lower median CMV
response magnitude than the controllers and HAART-sup-
pressed patients (P ? 0.01 and P ? 0.05, respectively). The
HIV-1 controllers, noncontrollers, and progressors did not dif-
fer in their responses to the HIV-1 peptide pool (P ? 0.05 for
all pairwise comparisons), but the controllers had stronger
HIV-1 pool responses than the HAART-suppressed patients
(P ? 0.05) (see Fig. S1B). Consistent with previous reports
using the IFN-? ELISPOT assay (2), there was no correlation
between the magnitude of the HIV-1 pool response and HIV-1
plasma viremia in untreated subjects (r2? 0.007, P ? 0.525)
(see Fig. S1C). There was also no significant association be-
tween the magnitude of the HIV-1 pool responses and the
HERV responses in untreated patients (r2? 0.033, P ? 0.164)
(see Fig. S1D).
HERV-specific immunity correlates with control of HIV-1.
Twenty-seven out of the 28 HERV peptides tested elicited at
least one positive response in the HIV-1-infected cohort, with
the majority of the peptides eliciting responses from two or
more individuals (Fig. 2). There was no difference between the
number of “unique” HERV peptides that stimulated a re-
sponse and the number of immunogenic HERV peptides that
shared four or more amino acids in common with an HIV-1
epitope (“similar”; median number of responses elicited by
both unique and similar HERV peptides, 4; P ? 0.63). In the
untreated HIV-1-infected individuals (controllers, noncon-
trollers, and progressors), the HIV-1 viral load was inversely
correlated with the magnitude of the HERV T cell response
(r2? 0.197, P ? 0.0002 by linear regression; Spearman r ?
?0.535, P ? 0.0001) (Fig. 3A) and the breadth of HERV T cell
FIG. 3. Both the magnitude (A) and the breadth (B) of the HERV
response were significantly inversely correlated with HIV-1 viral load
in untreated HIV-1-infected subjects. The HERV response was also
positively associated with higher CD4?T cell counts (C). r2and P
values (by linear regression) are as shown on the figure.
FIG. 2. The number of positive responses (defined as having a
magnitude of ?50 SFU/million PBMC and ?2 times the magnitude of
the negative control after subtraction of the value of the negative
control in IFN-? ELISPOT assays) to each HERV peptide among all
6980SENGUPTA ET AL.J. VIROL.
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response (r2? 0.115, P ? 0.005 by linear regression; Spearman
r ? ?0.339, P ? 0.005) (Fig. 3B). There was also a positive
correlation between HERV response magnitude and CD4?T
cell count in untreated patients (r2? 0.072, P ? 0.027 by linear
regression; Spearman r ? 0.269, P ? 0.027) (Fig. 3C). The
inverse correlation between HERV response magnitude and
HIV-1 viral load persisted even when the progressor group
(CD4?T cell count of ?250 cells/mm3) was excluded (r2?
0.091, P ? 0.037 by linear regression; Spearman r ? ?0.302,
P ? 0.037) (data not shown).
Mapping of responses against HERV-K Gag and Env in an
HIV-1-infected controller. In order to more comprehensively
characterize the T cell response against HERV-K (the young-
est and most transcriptionally active HERV family), we tested
a set of 336 overlapping 15-mer peptides spanning HERV-K
Gag and Env in a controller (P1, for whom large numbers of
PBMCs were available) who was infected with HIV-1 in 1990
and maintained an undetectable viral load over the following 2
decades. We detected a positive response to an HERV-K Env
peptide, CIDSTFNWQHRILLV, at a 2003 time point (Fig. 4)
that persisted and increased in magnitude over the next 5
Characterization of responses against two similar HERV-K
and HIV-1 epitopes in a unique subject. We further investi-
gated one particularly immunogenic peptide from the reverse
transcriptase region of HERV-K, FAFTIPAI (FI8). More than
one-quarter of the HIV-1-infected controllers that were tested
recognized this peptide. One individual (P2) had an especially
robust ELISPOT response (580 SPM) to FI8, which titrated to
1 ?g/ml (Fig. 5A). In addition, a strong response to an HIV-1
peptide with a similar sequence, TAFTIPSI (TI8), was ob-
served. This subject was infected with HIV-1 in 1999 and
maintained a CD4?T cell count above 400 cells/mm3and an
HIV-1 viral load below 6,000 copies/ml for almost a decade but
eventually began HAART in 2007 (Fig. 5B). Longitudinal
analysis of this individual’s responses to HERV-K FI8, HIV-1
TI8, and an HIV-1 peptide pool showed that all three waned
in 2005, just before the patient initiated HAART, whereas
the response to a CMV pp65 peptide pool was maintained
In order to determine the HLA restriction of the HERV-K
FI8 response in P2 (who was HLA-B51?), we tested the ability
of patient-derived PBMCs to recognize single HLA-allele B
cell transfectants that had been pulsed with peptide FI8 in an
ELISPOT assay. The peptide-pulsed HLA-B51?B cells were
recognized to a greater degree than the unpulsed B cells and
HLA-B51?B cells, confirming that P2’s response to this
epitope is restricted by HLA-B51 (Fig. 6).
The HERV-K FAFTIPAI response is more differentiated
and less activated than the HIV-1 TAFTIPSI response. To
further characterize P2’s responses to the two similar peptides,
HERV-K FI8 and HIV-1 TI8, we evaluated the functionality
and phenotypes of the CD8?T cell populations stimulated by
these antigens. We also included a CMV pp65 pool in order to
examine how these responses differ from one that is main-
tained long-term and is associated with a controlled chronic
viral infection. An intracellular cytokine detection assay was
used to analyze the simultaneous production of IFN-? and
TNF-? in antigen-specific cells. The CD8?population stimu-
lated by HERV-K FI8 had similar proportions of dual-cytokine
(IFN-? and TNF-?)-producing and monofunctional (IFN-??)
cells, as opposed to the primarily IFN-?-only-producing pop-
FIG. 4. Comprehensive mapping of responses to HERV-K Gag (A) and Env (B) in one HIV-1-infected elite controller (P1) using overlapping
peptides revealed a response to HERV-K Env peptide 104 (CIDSTFNWQHRILLV).
VOL. 85, 2011HERV-SPECIFIC T CELL RESPONSES IN HIV-1 INFECTION 6981
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ulation specific to the HIV-1 TI8 epitope (Fig. 7A; see also Fig.
S2A in the supplemental material).
To assess the differentiation stage of CD8?T cells that
produced cytokines against HERV-K FI8, HIV-TI8, and the
CMV pool, three subpopulations based on coexpression of T
cell maturation markers CD27 and CD28 were quantified.
CD8?T cells were defined as CD27?CD28?(early stage),
CD27?CD28?(intermediate stage), or CD27?CD28?(late
stage) (3, 5). The IFN-??population stimulated by HIV-1 TI8
had a higher proportion of cells at an early to intermediate
stage of differentiation (CD27?CD28?and CD27?CD28?).
In contrast, the IFN-?-producing cells stimulated by HERV-K
FI8 had largely a late-differentiation phenotype (CD27?
CD28?). Similarly, the IFN-??CMV-specific population had a
late stage of differentiation (Fig. 7B; see also Fig. S2B in the
supplemental material). In addition, the HERV-K FI8-specific
CD8?T cells were less activated (as measured by a CD38
mean fluorescence intensity [MFI] of 1,615) than the HIV-1
TI8-specific cells (CD38 MFI, 2,603) and had a level of acti-
vation that was comparable to that of the CMV-specific CD8?
population (CD38 MFI, 1,458) (Fig. 7C; see also Fig. S2C).
Single-tetramer staining of PBMCs from patient P2 and an
HIV-1 uninfected control with either HLA-B51-HERV-K FA
FTIPAI-APC (Fig. 8A) or B51-HIV-1 TAFTIPSI-PE (Fig. 8B)
showed tetramer-positive CD8?populations in P2 but not in
the control subject. Finally, by performing dual staining with
both tetramers, we confirmed that HERV-K FI8 and HIV-1
TI8 stimulated separate CD8?T cell populations in subject P2.
We identified two distinct populations that each stained posi-
tive for either the HLA B51-HERV-K FAFTIPAI-APC
tetramer or the B51-HIV-1 TAFTIPSI-PE tetramer (Fig. 8C).
Human endogenous retroviruses (HERVs) are thought to
be inactive elements of our genome, normally held in check by
host cellular restriction proteins such as APOBEC3G (11). We
hypothesized that the impairment of these controls in HIV-1
infection could expose the immune system to HERV antigens
that act as immutable targets for cytotoxic lysis, resulting in the
containment of HIV-1 viremia. Previously, our lab has shown
that HERV expression occurs in HIV-1-infected cells and that
HERV-specific CD8?T cell responses are stimulated in pri-
mary HIV-1 infection (12). These responses are negatively
correlated with HIV-1 viral load (12). Here, we extend these
observations to include individuals who have long-term chronic
HIV-1 infection, focusing on a rare subset of individuals who
are able to suppress HIV-1 indefinitely in the absence of com-
bination therapy. The mechanism of control in these HIV-1
controllers remains to be fully defined. Although there is evi-
dence that the HIV-specific T cell response contributes to
virologic containment (1, 10), it is clear from a number of
studies that other factors are likely to be involved (10, 26). A
recent genome-wide association study in a multiethnic cohort
of HIV-1 controllers and progressors showed that differences
FIG. 5. (A) The strength of subject P2’s T cell response to the
HERV-K FAFTIPAI peptide decreased according to the peptide con-
centration in the ELISPOT assay well. (B) Subject P2’s HIV-1 viral
load and CD4?T cell count over the course of 10 years. (C) This
individual’s response to HERV-K FI8, homologous HIV-1 peptide
TI8, and an HIV-1 peptide pool waned just before the subject was put
on HAART. In contrast, the CMV pp65 response was maintained over
these time points. Note that the response to HIV-1 TAFTIPSI was not
tested at the 2005 time point.
FIG. 6. Subject P2’s response to peptide HERV-K FI8 was found
to be restricted by the HLA-B51 allele in an ELISPOT-based assay
using peptide-pulsed and unpulsed HLA-matched and-mismatched B
cells as antigen presenting cells. P2’s PBMCs were incubated with the
following: lane 1, medium; lane 2, HERV-K FI8 peptide; lane 3,
HLA-B51?B cells pulsed with FI8 peptide; lane 4, HLA-B51?B cells
not pulsed with peptide; lane 5, HLA-B51?B cells pulsed with FI8
peptide; lane 6, HLA-B51?B cells not pulsed with peptide. Lane 7
represents all of the above conditions using B cells alone, without
subject P2’s PBMCs, in the ELISPOT assay.
6982SENGUPTA ET AL. J. VIROL.
on March 29, 2012 by guest
in HLA alleles explain 19% of the variance of host control
(27). In order to determine what effect, if any, HERV-specific
T cell responses have on viral control, we studied a cohort of
HIV-1 controllers and compared their responses to those of
untreated virologic noncontrollers and immunologic progres-
sors, patients on HAART, and HIV-1-uninfected controls.
While it is likely that robust CD4?T cell help contributes to
strong CD8?T cell responses, we sought to rule out that it was
the primary cause of any observed differential responses to
HERV antigens. Therefore, to avoid a confounding effect of
generalized immune dysfunction due to a lack of CD4?T cell
help, we performed additional analyses that excluded the im-
munologic progressor group. Our finding that the magnitude
of CMV pp65-specific responses was similar in the controller,
HAART-suppressed, and noncontroller groups supports the
fulfillment of this criterion.
We found that in chronic HIV-1 infection, controllers have
HERV responses with higher magnitude and greater breadth
FIG. 7. (A) Of the cytokine-producing cells in the population of
CD8?T cells stimulated by the HERV-K FAFTIPAI (FI8) peptide,
the proportion producing both IFN-? and TNF-? was similar to that
producing IFN-? only, whereas the HIV-1 TAFTIPSI (TI8)-specific
population was dominated by IFN-? production. (B) The HERV-K
FI8-specific CD8?population had a differentiation profile similar to
that of the CMV pp65 pool-specific cells (the highest proportion being
CD27?CD28?, consistent with a late-differentiation stage) and dis-
tinct from that of the HIV-1 TI8-specific cells (most cells being
CD27?, consistent with a less mature stage). (C) The HERV-K FI8-
specific CD8?population had a lower level of activation than the
HIV-1 TI8-specific CD8?cells, as measured by CD38 mean fluores-
cence intensity. Values are means and standard errors of the means.
FIG. 8. Single staining with an HLA-B51 HERV-K FI8 tetramer
(A) and an HLA-B51 HIV-1 TI8 tetramer (B) in subject P2 and a
healthy HIV-1-negative control. (C) Dual tetramer staining revealed
two distinct CD8?T cell populations within subject P2.
VOL. 85, 2011 HERV-SPECIFIC T CELL RESPONSES IN HIV-1 INFECTION6983
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than patients with viral suppression on HAART, virologic non-
controllers, immunologic progressors, and HIV-1-negative
subjects. Interestingly, controllers who lack HLA alleles that
are associated with protection from HIV-1 disease progression
(HLA-B27 and -B57) constituted a large proportion of the
subjects with the strongest HERV responses, suggesting that
there may be an alternative mechanism of HIV control (such
as HERV-specific cytotoxic T cells) in these controllers. There
was no difference in the magnitude of the HIV-1-specific re-
sponses between the controllers, noncontrollers, and progres-
sors (as defined by IFN-? production) and no correlation be-
tween this measure and HIV-1 plasma viremia. These data are
consistent with other studies reporting that neither the magni-
tude nor the breadth of the HIV-1-specific CD8?T cell re-
sponse is associated with a difference in viral load (2). Of note,
there was also no significant correlation between the strength
of HERV-specific responses and HIV-1-specific responses in
untreated patients, suggesting that these are independent vari-
ables. In contrast, we discovered that the HERV-specific T cell
response magnitude was inversely correlated with HIV-1 viral
load (even when the immunologic progressors were excluded),
suggesting a potential role for these responses in the control of
HIV-1. There was also a positive correlation between HERV
response magnitude and CD4?T cell count in untreated indi-
viduals. The group with viral suppression on HAART re-
sponded to fewer HERV epitopes and had weaker responses,
lending support to the hypothesis that these responses are a
cause rather than a consequence of viral control.
Longitudinal analyses of HERV responses in two subjects
revealed interesting differences. One individual (P2) had a
strong, titratable response to the HERV-K FAFTIPAI
epitope. This person was able to contain HIV-1 replication to
low levels and maintain normal CD4?T cell counts for almost
a decade. Interestingly, the patient eventually initiated
HAART, and we observed a decline in the magnitude of this
person’s HERV response in the few years preceding this junc-
ture. In contrast, another subject (elite controller P1) with
more than 15 years of undetectable HIV-1 viral load and nor-
mal CD4?T cell count without HAART had an increase in his
response to a different HERV-K epitope over 5 years. These
data are generally consistent with our primary hypothesis that
the generation and maintenance of HERV-specific responses
may contribute to virus control.
To investigate possible mechanisms of protection of the
HERV-specific response, we took advantage of P2’s compara-
bly strong ELISPOT responses to two very similar HERV and
HIV-1 peptides (HERV-K FI8 and HIV-1 TI8). After defining
the HLA restriction (B51) of the HERV response, we com-
pared its functionality and phenotype to those of the HIV-1
epitope and a CMV pp65 peptide pool. We found that within
the HERV-K FI8-specific population, there were as many cells
producing two cytokines as there were producing just one. In
contrast, the HIV response was dominated by monofunctional
(IFN-?-secreting) cells. IFN-? secretion is a first-line antiviral
defense mechanism and promotes the expression of TNF-?
receptors on the cell surface, among other functions (28).
TNF-?, in turn, has broad, beneficial effects in protective im-
munity and can kill virally infected target cells by binding to the
cell surface receptor and triggering an apoptosis signaling cas-
cade (reviewed in reference 21). The combination of the two
cytokines is therefore critical for an effective immune response
and has been shown to clear hepatitis B from hepatocytes and
lymphocytic choriomeningitis virus (LCMV) from acutely in-
fected mice (14, 23). HIV-1-specific CD8?T cells that can
generate a multifunctional response have been linked to slower
disease progression, and polyfunctional T cell responses are
associated with chronic control of other viral infections such as
CMV, Epstein-Barr virus (EBV), and influenza virus (6). We
were surprised to find that even a HERV epitope that shares
close homology with an HIV-1 sequence (having six out of
eight amino acids in common) could trigger a distinct CD8?T
cell response. Given the inverse correlation between HIV-1
viral load and the HERV response in our cohort, we might
expect this pattern to hold true for the total HERV-specific
and HIV-1-specific CD8?T cell population in this individual
and others, though this remains to be tested.
The observed differences in functionality of the HERV and
HIV-1 responses in our subject hinted that there could also be
a divergence in their phenotypic parameters, leading us to
examine their differentiation and activation profiles. Dual
staining with the HLA-B51 tetramers confirmed that two dis-
tinct and specific T cell populations that correspond to each of
these antigens exist and also ruled out the potential of a cross-
reactive effect. In our previous report (12), we compared the
phenotype of CD8?T cells responding to a CMV pool and to
two unique HERV and HIV-1 peptides without a high degree
of sequence homology. Unlike the maturationally deficient
HIV-1-specific population, the HERV-specific CD8?T cells
were similar to the CMV-specific CD8?T cells in having a
terminally differentiated phenotype that is associated with im-
proved viral control (25). Our present study confirms this even
though the two HERV and HIV-1 epitopes were similar and
also demonstrates that the HERV response was comparable to
the CMV response in its level of activation. The HERV-spe-
cific population had a late-differentiation (CD27?CD28?) and
low-activation (as measured by CD38 MFI) phenotype, which
has previously been associated with greater cytotoxic activity
(3). In contrast, the HIV-1-specific population was more im-
mature and had a higher level of activation. A disparity in
activation level between HIV-1-specific cells and CMV-specific
cells within the same individual was also recently reported by
Barbour, et al. (5). A lack of CD38 on the surface of virus-
specific T cells has been correlated with control of infections,
including HIV-1 and EBV, and has been linked to polyfunc-
tional cytokine production, MIP-1? chemokine secretion, pro-
liferative capacity, and lack of exhaustion as measured by PD-1
expression (6). Conversely, HIV-specific CD8?T cells from
viremic patients tend to be CD38?PD-1?and monofunctional
(secreting only IFN-?) (6). While the combination of these
measures was not examined in the current study, the lack of
CD38 expression on the HERV-specific T cell population sug-
gests that this epitope stimulates cytotoxic T lymphocytes
(CTLs) with these favorable characteristics and warrants ad-
Another factor contributing to the ability of HERV-specific
CTLs to proliferate and kill target cells may be linked to their
level of avidity. As HERVs are encoded in the human genome
and therefore represent self-antigens, T cells directed against
them may bind to their cognate epitopes with low avidity. This
is consistent with the increased peptide concentrations re-
6984SENGUPTA ET AL.J. VIROL.
on March 29, 2012 by guest
quired to generate a response in many of our subjects. Low-
avidity T cells (which are more prone to tolerance induction
and are stimulated only with high antigen load) have been
shown to proliferate better than high-avidity T cells and to lack
their replicative defects (16). Therefore, HERV-specific CTLs
may play a role in the containment of HIV-1 due to a superior
ability to proliferate. Of note, we previously showed that de-
spite being directed against self-antigens, HERV-specific
CTLs are not impaired in their ability to kill cells pulsed with
their cognate peptide (12). Overall, our present study has iden-
tified phenotypic and functional traits of the HERV-specific
response that may confer an enhanced ability beyond that of
most HIV-specific CTLs to effectively target and lyse infected
cells. Our finding that the HIV-1 viral load was lower in pa-
tients with stronger and broader HERV responses provides
empirical evidence for this.
Our results constitute the discovery of a novel correlate of
immune protection from disease progression in chronic HIV-1
infection and have implications for both a better understand-
ing of HIV-1 pathogenesis and a potential new approach to
HIV-1 vaccines. While the primary advantage of a therapeutic
agent derived from HERVs is their conserved nature, features
associated with a superior cytotoxic ability offer an additional
benefit to the induction of HERV-specific responses. Our find-
ings show that individuals who can control HIV-1 in the ab-
sence of HAART have the greatest HERV responses. This
HERV-specific immunity is correlated with the containment of
HIV-1 replication and preservation of CD4?T cell count in
untreated subjects and provides strong support for the further
investigation of an HERV-based HIV-1 vaccine.
We thank the NIH Tetramer Core Facility for synthesis of the
HLA-B51 tetramers and Lewis Lanier (University of California, San
Francisco) for providing single-HLA allele transfectant B cells used for
HLA restriction experiments. We acknowledge Neil Sheppard, Peter
Loudon, and James Merson of Pfizer for their helpful discussions.
This work was supported by the following funding sources: a Pfizer-
sponsored research agreement; NIH grants AI76059 and AI84113;
University of California, San Francisco-Gladstone Institute of Virol-
ogy and Immunology Center for AIDS Research (CFAR), an NIH-
funded program (P30 AI027763); and The Fogarty International Cen-
ter, grant D43 TW00003. This work was supported in part by the
Centers for AIDS Research at UCSF (PO AI27763) and the UCSF
Clinical and Translational Science Institute (UL1 RR024131). Addi-
tional support was provided by NIAID (RO1 AI087145 and
K24AI069994), American Foundation for AIDS Research (106710-40-
RGRL), the NIH/NIAID CFAR Network of Integrated Clinical Sys-
tems (grant 1 R24 AI067039-1), and the Ragon Institute. C.E.O.
was funded by Consejo Nacional de Ciencia y Tecnología, Mexico
R.B.J., K.E.G., D.F.N., F.M.H., and M.A.O. are listed as inventors
on a patent application related to this work.
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