Journal of Immunological Methods 365 (2011) 27–37

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Journal of Immunological Methods

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Research paper

Development of a HIV-1 lipopeptide antigen pulsed therapeutic dendriticcell vaccine

Amanda Cobb a,b, Lee K. Roberts c, A. Karolina Palucka a, Holly Mead a, Monica Montes a,Rajaram Ranganathan a, Susan Burkeholder a, Jennifer P. Finholt a, Derek Blankenship a,Bryan King d, Louis Sloan d, A. Carson Harrod a,b, Yves Lévy e,f,g,⁎,1, Jacques Banchereau a,⁎,1

a Baylor Institute for Immunology Research, Dallas, TX, INSERM U899, United Statesb Baylor University, Waco, TX, United Statesc Roberts Biotech Consulting, LLC, Cordova, TN, United Statesd North Texas Infectious Diseases Consultants, Dallas, TX, United Statese INSERM U955, Créteil, F-94010, Francef Université Paris Est Créteil, Faculté de Médecine, Créteil, F-94010, Franceg AP-HP, Groupe Henri-Mondor Albert-Chenevier, Immunologie clinique, Créteil, F-94010 France, French National Agency for AIDS Research (ANRS) Paris, France

a r t i c l e i n f o

Abbreviations: AIDS, acquired immunodeficiency vResearch); APC, antigen presenting cells; CFSE, carboxynon-essential amino acids, 1% L-glutamine, and 0.1%positive; CTL, cytotoxic CD8+ T-cells; DC, dendriticgranulocyte–macrophage colony-stimulating factor; Htype-1; IFN, interferon; IL, interleukin; IONO, ionompeptides that are covalently linked at their C-terminamixed lymphocyte reaction; PBMC, peripheral blood m⁎ Corresponding authors. Lévy is to be contacted a

Créteil, F-94010, France. Tel.: +33 1 49 81 44 42; fax75204, United States. Tel.: +1 214 820 7450; fax: +

E-mail addresses: yves.levy@hmn.aphp.fr (Y. Lévy)1 On behalf of the ANRS HIV Vaccine Network.

0022-1759/$ – see front matter © 2010 Published bydoi:10.1016/j.jim.2010.11.002

a b s t r a c t

Article history:Received 28 October 2010Accepted 5 November 2010Available online 18 November 2010

In the search for a therapeutic HIV-1 vaccine, we describe herein the development of amonocyte-derived dendritic cell (DC) vaccine loaded with a mixture of HIV-1-antigenlipopeptides (ANRS HIV-LIPO-5 Vaccine). LIPO-5 is comprised of five HIV-1-antigen peptides(Gag17–35, Gag253–284, Nef66–97, Nef116–145, and Pol325–355), each covalently linked to apalmitoyl-lysylamide moiety. Monocytes enriched from HIV-1-infected highly active anti-retroviral therapy (HAART)-treated patients were cultured for three days with granulocyte–macrophage colony-stimulating factor and alpha-interferon. At day 2, the DCs were loadedwith ANRS HIV-LIPO-5 vaccine, activated with lipopolysaccharide, harvested at day 3 andfrozen. Flow cytometry analysis of thawed DC vaccines showed expression of DC differentiationmarkers: CD1b/c, CD14, HLA-DR, CD11c, co-stimulatory molecule CD80 and DC maturationmarker CD83. DCs were capable of eliciting an HIV-1-antigen-specific response, as measured byexpansion of autologous CD4+ and CD8+ T-cells. The expanded T-cells secreted gamma-IFNand interleukin (IL)-13, but not IL-10. The safety and immunogenicity of this DC vaccine arebeing evaluated in a Phase I/II clinical trial in chronically HIV-1-infected patients on HAART(clinicaltrials.gov identifier: NCT00796770).

© 2010 Published by Elsevier B.V.

Keywords:DCANRS-LIPO-5 vaccineHIVVaccine developmentAntigen-specific T-cellsIFN-γ

irus; AA, amino acids; ANRS, Agence Nationale de Recherche sur le SIDA (French National Agency for AIDSfluorescein succinimidyl ester; cRPMI, complete RPMI 1640with 2.5% hepes, 1% penicillin/streptomycin, 1%β-mercaptoethanol, supplemented with 10% human serum type AB; C−, control negative; C+, controlcells; DMSO, dimethyl sulfoxide; FACS, fluorescence-activated cell sorting (flow cytometry); GM–CSF,AART, highly active antiretroviral therapy; HC, HIV-1 controllers; HIV-1, human immunodeficiency virusycin; LIPO-5, ANRS HIV-1-antigen lipopeptides: Gag17–35, Gag253–284, Nef66–97, Nef116–145, and Pol325–355l end to a palmitoyl-lysylamide moiety; LPS, lipopolysaccharide; LTNP, long-term non-progressors; MLR,ononuclear cells; PMA, phorbol 12-myristate 13-acetate; rhCD40L, recombinant-human CD40 ligand.

t Groupe Henri-Mondor Albert-Chenevier, Immunologie clinique, 51 Av Maréchal de Lattre de Tassigny,: +33 1 49 81 24 69. Banchereau, Baylor Institute for Immunology Research, 3434 Live Oak, Dallas, TX1 214 820 4813., jacquesb@baylorhealth.edu (J. Banchereau).

Elsevier B.V.

28 A. Cobb et al. / Journal of Immunological Methods 365 (2011) 27–37

1. Introduction

In the last decade, the advent of highly active antiretroviraltherapies (HAART) that containHIV-1 replication has changedthe course of HIV-1 infection by reducing the AIDS-relatedmorbidity and mortality of patients (Virgin and Walker,2010). This clinical benefit is clearly related to the limitationof the immunological damage that is caused by HIV-1replication as well as to the restoration of CD4+ T-cell countsand specific responses against pathogens (Kaufmann et al.,2000; McMichael et al., 2010). However, HAART induces alarge range of toxicities, raising the concern of long-term useover decades. Therefore, the development of therapeuticstrategies that may help to control viral replication and tolimit drug exposure is essential.

The rationale for therapeutic immunization in HIV-1infection is based on several lines of evidence suggestingthat the immune system contributes to the long-term controlofHIV-1 replication (Borrowet al., 1994; Koup et al., 1994; Caoet al., 1995; Rosenberg et al., 1997; Deeks and Walker, 2007).Remarkably, a state of durable evolution of HIV-1 infectionwithout a significant decrease of CD4+ T-cell counts and/ordetectable viral replication does occur in a limited number ofuntreated patients called Long-Term Non-Progressors (LTNP,5–15% of HIV-infected patients) (Cao et al., 1995) or HIV-1controllers (HC; less than 1%) (reviewed in Deeks andWalker,2007). These clinical observations provide clear evidence thatdurable containment of HIV-1 replication and/or preventionof disease progression without antiretroviral therapy arepossible. However, continued viral replication leading toprogressive immune destruction is the rule in a majority ofpatients. In the long term, although HIV-1-specific CD4+ andCD8+ T-cells may be detectable in patients at different timepoints of the disease, these cells are functionally impaired andfail to control viral replication after treatment discontinuation(Appay et al., 2000; Goepfert et al., 2000; Carcelain et al., 2001;Champagne et al., 2001). Therefore, the hope is that animmune and/or vaccine intervention might mobilize some ofthe mechanisms that mediate control of viral replication inthese rare patients and, by doing so, control viral replication orlower the viral “set point” in patients who did not achieve thisequilibrium on their own.

Studies in macaques have indeed shown that vaccinesinducing CD8+ T-cells can result in a dramatic decrease ofserum simian immunodeficiency virus levels (Liu et al., 2009;Bonaldo et al., 2010). When applied in humans, this approachcould reduce patient-induced viral spreading and notnecessitate HAART treatment. In designing a therapeuticvaccine for treating HIV/AIDS, an important aim is to increasethe number and efficacy of the polyfunctional HIV-1 antigen-specific cytotoxic CD8+ T-cells (CTL), CD4+ T-cell help, andNK lysing (McMichael et al., 2010). The expansion of thispolyfunctional CTL population would, thus, mimic the immu-nological status seen in LTNP.

Some of the difficulty in developing an effective vaccineagainst HIV-1 lies in the fact that HIV-1 virus is highlymutated upon rapid replication within an individual patient,thus expanding the number of antigens required for devel-oping a universal vaccine (McMichael et al., 2010). Since1994, the French National Agency for AIDS Research (ANRS)has studied conserved regions of HIV-1 for use as antigens in

immunotherapeutic vaccine clinical trials. Of these regions, fiveimmunogenic peptides ranging from 19 to 32 amino acids inlength were identified for inclusion in the ANRS HIV-LIPO-5vaccine. The ANRS HIV-LIPO-5 vaccine is comprised of theGag17–35, Gag253–284, Nef66–97, Nef116–145, and Pol325–355 pep-tides that are covalently linked at their C-terminal ends to apalmitoyl-lysylamide moiety (Levy et al., 2005; Durier et al.,2006). Clinical trials in healthy volunteers and chronically HIV-1-infected patients demonstrated immunogenicity of LIPO-5(Pialoux et al., 2001; Goujard et al., 2007; Launay et al., 2007;Pialoux et al., 2008; Salmon-Ceron et al., 2010).

Dendritic cells (DC) are potent antigen presenting cells(APC) capable of inducing CTL and helper T-cell responses thatare essential in the process of vaccination (Banchereau andSteinman, 1998; SteinmanandBanchereau, 2007;Alvarez et al.,2008; Yu et al., 2008; Sabado and Bhardwaj, 2010). Severalgroups have demonstrated the efficacy of DC vaccines in thetherapeutic treatment of cancer and viral infections includingHIV-1 (Timmerman and Levy, 2000; Banchereau et al., 2005;Palucka et al., 2007;Melief, 2008).Herein,we report on the pre-clinical studies that were undertaken to develop a therapeuticDC vaccine by culturing monocytes with a combination ofgranulocyte -macrophage colony-stimulating factor (GM-CSF)/alpha-interferon (IFN-α), loading them with LIPO-5 andactivation with lipopolysaccharide (LPS).

2. Patients and methods

2.1. Patient population

Between October 2007 and December 2008 leukapheresiswas collected at the Baylor University Medical CenterApheresis Collection Center (Dallas, TX) from eight HIV-1-infected adult patients who were on the HAART regimen(identified as A1–A8) and eight healthy, non-HIV-1-infectedadult volunteers (identified as H1–H8; SupplementaryTable 1). The HIV-1 patients who volunteered for this studywere selected according to the following criteria: plasmaHIV-1 RNA viral load level b50 copies/mL and CD4+ T-cell countsN500 cells/mm3 of peripheral blood. The HIV-1-infectedpatients were recruited at the North Texas Infectious DiseasesConsultants in Dallas, TX. The study was approved by theInstitutional Review Board of the Baylor Health Care System(Dallas, TX) and informed consent was obtained from allindividuals participating in the study.

2.2. Monocyte enrichment

Peripheral blood monocytes were enriched from theleukapheresis according to cellular density and size byelutriation (Elutra™, CaridianBCT, Lakewood, CO) as per themanufacturer's recommendations. The Elutra's automatedprogram separated the cells into five fractions using variousflowrates andcentrifuge speeds. Elutriation Fraction5 consistedmainly of monocytes (~85% on average), with the remainder ofthe cells being granulocytes, lymphocytes, eosinophils andbasophils, as measured by diagnostic hemacytometry ABXPentra 60C+ (Horiba ABX Diagnostics, Montpellier, France).This instrument incorporates cytochemistry, focused flowimpedance, light absorbance and flow cytometry for calculatingcomplete blood cell count with differential. Cell counts and

29A. Cobb et al. / Journal of Immunological Methods 365 (2011) 27–37

purity of subsequent elutriated fractions were also assessed byABX Pentra 60C+. The Fraction 5 of elutriated cells was used asthe source of monocytes for production of the DC vaccine.

2.3. DC vaccine production

On day 0 of the process, the elutriated monocytes wereresuspended in serum-free CellGro® DC culture media(CellGenix Technologie Transfer GmbH, Germany) at aconcentration of 1×106 cells/mL for culture in disposableplastic culture bags (AFC, Gaithersburg, MD). The media weresupplemented with 100 ng/mL granulocyte-macrophagecolony-stimulating factor (GM-CSF) (Leukine®, Berlex,Wayne, NJ) and 500 IU/mL alpha-interferon (IFN-α) (IntronA®, IFN-α-2b, Merck/Schering-Plough, Kenilworth, NJ).After 24 h in culture, fresh cytokines were added. On day 2,LIPO-5 was added at various concentrations, over a range of0.003 μM to 0.3 μMper peptide. On day 3 of culture, 6 h prior tocell harvest, lipopolysaccharide (LPS; NIH, Bethesda, MD) wasadded to the cell suspension at 5 EU/mL to activate the LIPO-5-loaded DC. After LPS activation, the DC were harvested, washedwith normal saline (0.9% NaCl, USP grade; Hospira, Lake Forest,IL), and suspended at 30×106viable cells/mL in freezing-solution for filling into glass vaccine vials. The DC vaccinefreezing-solution consisted of 80% heat-inactivated autologousserum, 10% Plasma-Lyte A (Hospira) supplemented with 5%dextrose (Baxter, Deerfield, IL) and 10% dimethyl sulfoxide(DMSO) (Cryoserv®, Bioniche, Lake Forest, IL). The cells werefrozen in a rate-controlled freezer and the vials were stored at−180 °C in the vapor-phase of a liquid nitrogen tank.

The DC vaccines were thawed and diluted 10-fold withnormal saline. For culture, cells were centrifuged andresuspended in cRPMI medium (RPMI 1640 with 2.5%HEPES, 1% penicillin/streptomycin, 1% non-essential aminoacids, 1% L-glutamine, and 0.1% β-mercaptoethanol (Invitro-gen, Carlsbad, CA) supplementedwith 10% human serum typeAB (Gemini Bio-Products, West Sacramento, CA, US)).

2.4. HIV-1 antigen peptides

The ANRS-HIV LIPO-5 vaccine is a mixture of five HIV-1-antigen lipopeptides. The five HIV-1-antigen peptides are:Gag17–35 (EKIRLRPGGKKKYKLKHIV K(Palm)-NH2), Gag253–284(NPPIPVGEIYKRWIILGLNKIVRMYSPTSILD K(Palm)-NH2),Nef66–97 (VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGL K(Palm)-NH2), Nef116–145 (HTQGYFPDWQNYTPGPGV-RYPLTFGWLYKL K(Palm)-NH2), and Pol325–355 (AIFQSSMTKI-LEPFRKQNPDIVIYQYMDDLY K(Palm)-NH2). Each peptide wasmodified in the C-terminal position with an N-ε-palmitoyl-lysylamide group. The LIPO-5 vaccine, supplied as a lyophilizedmixture of 500 μg of each lipopeptide (2.5 mg of lipopeptide/vial), was dissolved in 5% dextrose (USP grade, Hospira)injectable solution to a stock concentration of approximately134 μMper lipopeptide. The sameHIV-1-antigen peptides (thatis, peptides with the identical sequence to those in LIPO-5, butwithout the palmitoyl-lysylamide moiety; ‘non-lipidated pep-tides’) were used in the autologous T-cell response assay. Thestock concentration of the non-lipidated peptides was 10 mMper peptide suspended in DMSO. Overlapping 11 amino acid(AA) peptide libraries (15 AA in total length per peptide) of theHIV-1 Gag and Nef proteins were used to define the HIV-1-

antigen epitopes. The peptide library stock concentration was10 mM per peptide in an acetonitrile (Fluka, Buchs, Germany)solution.

2.5. Phenotypic characterization

The phenotypes of the DC vaccines were assessed usingfluorescence-conjugated monoclonal antibodies: CD14-PE,HLA-DR-PE, CD11c-APC, CD80-PE, CD83-PE, CD207-PE (IOTest & Validation, Bailey, Roxburghshire, UK), and CD1b/c-FITC (Biosource, Carlsbad, CA). Cell staining profiles wereacquired on a FACSCalibur™ Flow Cytometer (BD Bioscience,San Jose, CA) and analyzed with FlowJo software (Treestar,San Carlos, CA).

2.6. DC cytokine and chemokine secretion assay

The cytokine and chemokine secretion profile of the DCvaccines were assessed by culturing 200,000 thawed DC sus-pended in cRPMImedium in 96-well microtiter plates. rhCD40L(R&D Systems, Minneapolis, MN) at 100 ng/mL was added toselect wells. The supernatant was harvested after 48 h followedby analysis using cytokine multiplex assays. The followingcytokines and chemokines were analyzed: IL-1β, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, IP-10, MCP1, MIP1α, Rantes, and TNFα.

2.7. Mixed lymphocyte reaction (MLR)

The potency of the DC vaccine was assessed by using anallogeneic T-cell proliferation assay. Lymphocytes wereisolated by elutriation from apheresis blood product of ahealthy, non-HIV-1-infected patient and frozen. For the assay,lymphocytes were thawed, washed by centrifugation, stainedwith carboxyfluorescein succinimidyl ester (CFSE, Invitro-gen), and resuspended at a concentration of 1×106cells/mLin cRPMI medium. Using a 96-well plate, DC and lymphocyteswere added to designated wells for a final ratio of 1/20, 1/100,and 1/500, that is, 5000, 1000 and 200 DC to 105 lymphocytes,respectively. The negative (C−) and positive (C+) controlsfor this assay were lymphocytes cultured in media alone orwith CD3/CD28 Dynabeads (Invitrogen), respectively. Eachcontrol and test condition was cultured in triplicate. After fivedays of co-culture, the cells were harvested from the 96-wellplate, stained with anti-CD3 and anti-CD8 fluorescence-conjugated monoclonal antibodies (CD3-PerCP and CD8-APC from BD Bioscience) and analyzed by flow cytometry todetermine the percentage of dividing CD3+CD8+ T-cells andCD3+CD8− T-cells (CD4+ T-cells) based on reduced CFSEstaining intensity.

2.8. Autologous HIV-1-antigen-specific T-cell responses

Lymphocytes enriched in elutriation Fractions 2/3/4, andstored frozen, were isolated from the same HIV-1 patient'sleukapheresis that was used to produce the DC vaccine fromelutriation Fraction 5. The frozen lymphocytes were thawed,washed by centrifugation, and then resuspended at aconcentration of 1×106cells/mL in cRPMI medium. Autolo-gous DC vaccine and lymphocytes were co-cultured induplicate or triplicate, in a 24-well tissue culture plate at aratio of 1/20 (100,000 DC to 2×106 lymphocytes) and

Table 1Distribution of cells in elutriation Fraction 5 collected from HIV-1-infectedpatients and healthy donors.

Cell type Healthy donors HIV-1-infected patients

Monocytes 87.7±4.2 a 85.0±8.4Neutrophils 2.8±3.1 5.8±10.3Lymphocytes 8.3±3.2 7.9±3.7Eosinophils 0.1±0.2 0.2±0.3Basophils 1.1±0.6 1.1±0.3

a Data are presented as the mean±standard deviation of the percentageof each cell type present in elutriation Fraction 5 collected from the 8 healthydonors and 8 HIV-1-infected patients.

30 A. Cobb et al. / Journal of Immunological Methods 365 (2011) 27–37

incubated for a total of 10 days. 10 IU/mL IL-7 (R&D Systems)was added to cultures. On day 2, 100 IU/mL IL-2 (Aldesleukin,Proleukin®; Bayer Healthcare and Novartis, Emeryville, CA)was added. To assess the specificity of the T-cell responseelicited by the DC vaccines, lymphocytes were harvested atday 10 and were restimulated with individual non-lipidatedHIV-1-antigen peptides for 4 h. Each DC-vaccine-stimulatedlymphocyte condition was restimulated without peptides(background control, C−) or with PMA 2 ng/mL andionomycin 1 μM (PMA/IONO; both from Sigma-Aldrich, StLouis, USA) as a gamma-IFN (IFN-γ)-positive control (C+).After 1 h of restimulation, BD Golgistop™ was added to blockany further protein transport. After a total of 4 h, the cellswere harvested and cell surface (CD3 and CD8) andintracellular (IFN-γ) staining was performed.

2.9. Epitope mapping of the autologous T-cell response

Autologous lymphocytes from HIV-1 patients A7 and A8were culturedwith LIPO-5-loadedDC vaccine for 10 days. As a

Fig. 1. Schematic representation of DC vaccine manufacturing. (A) Collection of PBMC(C) Enriched monocytes are cultured with GM–CSF and IFN-α, loaded with LIPO-5

control, PBMC collected from HIV-1 patients A7 and A8 werecultured for 7 days in media containing individual 15 AApeptides at 10 μM from HIV-1 proteins Gag and Nef peptidelibraries. At day 2, 100 IU/mL IL-2 was added. At the end ofthe “antigen-priming” culture, the DC vaccine expandedlymphocytes and the peptide-library-stimulated PBMC wereharvested, washed and dispensed into the wells of a 96-wellmicrotiter plate. To eachwell, one of the peptides from theHIVGag and Nef peptide libraries was added to the cell culturemedia at 10 μM. The cells in each well were restimulated withone of the individual 15 AA HIV-1-antigen peptides for 48 h.Supernatants were collected and cytokine multiplex assayswere employed to analyze IFN-γ, IL-10, and IL-13.

3. Results

3.1. Generation of DC from enriched monocytes

The purity of the monocytes collected in elutriationFraction 5 was comparable between HIV-1-infected patients(85.0±8.4%) and healthy donors (87.7±4.2%) (Table 1).Likewise, the percentage of elutriation Fraction 5 lympho-cytes, neutrophils, eosinophils and basophils were similarbetween the two donor groups (Table 1). The total number ofmonocytes isolated from HIV patients (22.78±9.26×108)was higher but not significantly different (T-test, pb0.079)than that of healthy donors (15.33±6.17×108).

Antigen-loaded DC vaccines were manufactured in three-day monocyte culture with GM–CSF and IFN-α, as describedin Patients and methods and illustrated in Fig. 1. Thawed DCvaccines were used to conduct the characterization assaysdescribed in the following discussion. The phenotype of atypical DC vaccine manufactured with monocytes from an

by leukapheresis. (B) Leukapheresis is separated by cellular density and size(0.1 μM), activated with LPS, (D) harvested and frozen.

.

Fig. 2. LIPO-5-loaded DC vaccine phenotype. FACS analysis dot-plots of the phenotype of ANRS HIV-LIPO-5-loaded DC vaccine from HIV-1 patient A4. DCs werethawed, washed, stained with various fluorescence-conjugated monoclonal antibodies, acquired on FACS Calibur, and analyzed by FlowJo Software.

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HIV-1-infected patient (A4) is shown in Fig. 2 and resultsobtained with patients A1–A6 are summarized in Table 2. TheDC vaccines had a high frequency of HLA-DR+/CD11c+

(mean, 94%; range, 88–98%) and CD80+ cells (mean, 95%;range, 91–97%). Most DCs expressed CD1b/c+ (mean, 62%;range, 36–86%), CD14+ (mean, 62%; range, 17–87%), andCD83+ (mean, 74%; range, 32–96%). A fraction of cellsexpressed CD207+ (Langerin; mean, 28%; range, 7–46%).Thus, the DC vaccines express surface molecules character-istic of DC with co-stimulatory molecules necessary for T-cellinteraction.

Table 2Phenotypes of DC vaccines prepared with monocytes from HIV-1-infectedpatients A1–A6.

DCvaccine

HLA-DR+/CD11c+

CD1b/c+ CD14+ CD80+ CD83+ CD207+

A1 94 a 60 17 96 95 19A2 96 62 39 97 96 27A3 88 36 67 96 77 7A4 98 67 84 92 77 46A5 98 86 76 96 32 42A6 91 58 87 91 68 28Mean 94.2 61.5 61.6 94.8 74.2 28.3Std dev 3.9 16.0 27.8 2.5 23.5 14.5

a Values represent the percentage of cells in the DC vaccine that expressthe specified cell surface marker(s).

3.2. HIV-DC vaccines secrete pro-inflammatory cytokines andchemokines

We analyzed the cytokines and chemokines secretedwithin 48 h by HIV-DC vaccines either spontaneously(Table 3) or after CD40 ligation (Supplementary Table 2).The DC vaccines generated from three patients secretespontaneously high levels of pro-inflammatory cytokines,IL-1β (660–4000 pg/mL), IL-6 (3300–28,000 pg/mL), and IL-8 (4600–5100 pg/mL). IL-10 was also secreted in lowamounts ranging from 40 to 300 pg/mL. DCs from 2 of the 3patients were found to secrete IL-12p40 (300–1500 pg/mL)and IL-12p70 (30–170 pg/mL). TNFαwas also secreted (20 to630 pg/mL). The DCs secreted a panel of chemokines,including MCP-1 (CCL2, 480–5500 pg/mL), MIP1α (CCL3,3700–4500 pg/mL), Rantes (CCL5, 710–1120 pg/mL), and theinterferon-inducible chemokine IP-10 (CXCL10, 1800–11,400 pg/mL). In one analyzed patient, the addition ofsoluble rhCD40L to the DC vaccine culture resulted only inmoderate if any enhancement of cytokine or chemokinesecretion (Supplementary Table 2). Thus, the DC vaccinedemonstrates features of activated DCs.

3.3. The DC vaccine elicits the proliferation of allogeneic T-cells

Allogeneic T-cells collected from normal healthy donorswere stained with CFSE. One hundred thousand lymphocytes

Table 3LIPO-5-loaded DC vaccines secrete pro-inflammatory cytokines and chemotactic chemokines of patients A1–A3. Two hundred thousand DCwere cultured for 48 hwith no additional signaling (patients A1–A3).

Patient Cytokines Chemokines

IL-1β IL-6 IL-8 IL-10 IL-12p40 IL-12p70 TNFα MCP1 MIP1α Rantes IP-10

A1 658 a 3326 4951 38 1 18 20 481 3703 712 1804A2 746 14,936 4597 104 307 29 109 1073 4500 1120 8974A3 3991 27,978 5094 293 1510 165 626 5497 4500 952 11,420

a Data are presented as pg/mL.

Fig. 3. LIPO-5-loaded DC vaccine induces allogeneic T-cell proliferation. Function of LIPO-5-loaded DC vaccine from HIV-1 patient A3 in allogeneic MLR asmeasured by CFSE dilution. DC were washed of DMSO, counted and resuspended in cRPMI+10% human serum type AB. DC and CFSE-stained lymphocyteswere cultured at ratios of 1/20, 1/100, or 1/500, DC/lymphocytes. After 5 days, the cells were stained with CD3, CD8 and analyzed by flow cytometry. Controls wereT-cells with media alone (C−) or with CD3/CD28 dynal beads (C+).

32 A. Cobb et al. / Journal of Immunological Methods 365 (2011) 27–37

were culturedwith the DC vaccine added at various ratios from1/20 to 1/500. After 5 days, the cells were stained withfluorescence-conjugated anti-CD3 (PerCP) and anti-CD8(APC) and analyzed by flow cytometry. Fig. 3 represents adot-plot analysis of the MLR assay performed with the DCvaccine from HIV-1 patient A3. The cells are first gated on theCD3+ T-cell population and further analyzed by assessing CFSEstaining intensity in the CD8+ and CD8− T-cells. Table 4 reportsthe allogeneic T-cell responses of the total CD3+ population forHIV-1 patientDC vaccines (A1–A6). At a ratio of 1/20, amean of43% of CD3+ T-cells diluted CFSE (range, 13–85%). At a ratio of1/100, 20% of CD3+ T-cells diluted CFSE (range, 7–36%). At aratio of 1/500, 4% of CD3+ T-cells diluted CFSE (range, 0.2–8%).The proliferation background (C−) was 0.9% (range, 0.1–1.6%).CD3/CD28 dynal beads were used as a positive control, and72% of CD3+ T-cells diluted CFSE (range, 56–88%). Thus,monocyte-derived GM–CSF/IFN-α, ANRS HIV-LIPO-5 loaded,

Table 4Allogeneic MLR summary of HIV-1 vaccines A1–A6. Values are presented aspercentage (%) CD3+CFSElo T-cells.

DC vaccine 1/20 1/100 1/500 C− C+

A1 50.0 21.0 0.2 0.1 72.0A2 85.0 31.7 3.8 1.6 87.8A3 65.0 36.0 8.0 0.3 88.0A4 25.0 14.0 6.0 0.6 70.4A5 18.3 10.7 4.9 1.0 55.6A6 12.6 7.1 3.5 0.4 56.4Mean 42.8 20.3 4.4 0.9 71.7Std dev 29.0 12.0 2.6 0.6 14.3

Fig. 4. IFN-γ antigen-specific responses peak at 0.1 μM/peptide. DC vaccinesloaded with LIPO-5 concentrations ranging from 0.003 μM to 3.0 μM wereprepared with monocytes obtained from HIV-1 patient A5. The assayconsisted of co-culturing autologous lymphocytes with the different DCvaccines for 10 days, followed by a 4 h restimulation with the HIV-1-antigennon-lipidated peptides. FACS analysis was used to determine the percentageof IFN-γ-producing CD8+ T-cells responding to the Gag17–35 (Gag 17—gray),Nef66–97 (Nef 66—diagonal stripes), and mixture of the five HIV-1-antigenpeptides (Mix-black). The negative control is the response of T-cells thatwereco-cultured with DC that were not loaded with any HIV-1-antigen peptides.

LPS-activated DC vaccines generated fromHIV-1 patients act aspotent antigen presenting cells in the allogeneic MLR.

3.4. The DC vaccine induces HIV-1-antigen-specific CD4+ andCD8+ T-cell responses

To determine the dose of LIPO-5 required to load the DC,several batches of DC vaccines were loaded using a range of

33A. Cobb et al. / Journal of Immunological Methods 365 (2011) 27–37

LIPO-5 concentrations (from 0.003 μM to 3.0 μM). Thesedifferentially loaded DC vaccines were then analyzedfor their ability to expand HIV-1-antigen-specific autologousT-cells in a ten-day co-culture assay illustrated in Supple-mentary Fig. 1A. The expanded cells were then restimulatedfor 4 h with the HIV-1-antigen peptides to assess thepercentage of responding IFN-γ-producing T-cells byflow cytometry (Fig. 4). The peak responses were observedwith T-cells that had been co-cultured with DC loaded atLIPO-5 concentrations≥0.1 μM. Because this finding wasconsistent across DC vaccine batches (data not shown), theLIPO-5 loading concentration was set at 0.1 μM for manufac-ture of the HIV-1 therapeutic DC vaccine product targeted forclinical evaluation.

We then evaluated six clinical-grade DC vaccines fromHIV-1-infected patients for the ability to elicit HIV-1-antigen-

specific T-cell responses. One hundred thousand LIPO-5(0.1 μM)-loaded DC were cultured with 2×106 autologouspatient lymphocytes for 10 days in the presence of IL-2 andIL-7. The expanded cells were then restimulated for 4 h withthe five individual HIV-1 peptides (10 μM each, no lipidatedtail) to assess the percentage of IFN-γ-producing T-cells byflowcytometry. A fraction of the expanded T-cells were culturedwithout peptides to assess background IFN-γ secretion or withPMA/IONO to assess potential IFN-γ secretion and the validityof the assay.

Comprehensive scatter-graphs and statistical analysis ofthe antigen-specificity are shown in Fig. 5. The analysis beganwith gating on the viable lymphocyte population (Supple-mentary Fig. 2) followed by gating on CD3+ T-cells. Furtheranalysis of the gated CD3+ T-cells reveals that the specificHIV-1-antigen peptides are recognized by the responding

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Fig. 5. IFN-γ antigen-specific responses elicitedby LIPO-5-loadedDCvaccine.ANRSHIV-LIPO-5-loadedDCvaccines induceautologousT-cells to respond to specificHIV-1-antigen peptides. FACS analysis was performed to determine the percentage of DC-vaccine-expanded autologous T-cells that respond to the specific HIV-1-antigenpeptides in LIPO-5 (Gag17–35 [Gag 17], Gag253–284 [Gag 253], Nef66–97 [Nef 66], Nef116–145 [Nef 116] and Pol325–355 [Pol 325]) or to detect background by negative control(C−). Panel A, CD8+ and CD8− T-cells that produced IFN-γ were identified as responding to HIV-1-antigen peptides. The analysis was conducted with DC vaccineprepared fromsixHIV-1patients (A1–A6). Panel B reports the average percentage of CD3+CD8+IFN-γ+(left panel) andCD3+CD8-IFN-γ+(rightpanel) for patientsA1–A6. Positive peptide responses are identified as those greater than three standard deviations from themeanof the negative controls as indicated by an asterisk (*) abovethe dotted line.

34 A. Cobb et al. / Journal of Immunological Methods 365 (2011) 27–37

IFN-γ-producing CD8+ or CD4+ T-cells. A representative dot-plot analysis of single samples is illustrated in Fig. 5A. Thebackground IFN-γ-secreting CD8+ and CD4+ T-cells rangedfrom 0.03 to 0.2% and from 0 to 0.07%, respectively. When DCsare cultured without LIPO-5 and co-cultured with autologouslymphocytes in the same manner, IFN-γ secretion was notdetected (Fig. 4). IFN-γ secretion was seen when cells wereculturedwith PMA/IONO (data not shown). Fig. 5B reports thecomprehensive analysis of the average (duplicate or tripli-cate) percentage of CD3+CD8+IFN-γ+ andCD3+CD8−IFN-γ+

for patients A1–A6. Positive peptide responses are identifiedas those greater than three standard deviations from themeanof negative controls. CD8+ positive peptides had to be greaterthan 0.28% and CD4+ positive peptides had to be greaterthan 0.10%. Patient A1 displayed CD8+ T-cell responses toNef 66, Nef 116, and Pol 325. Patient A2 displayed CD8+ T-cellresponses to Nef 116 and CD4+ T-cell responses to Gag 253.Patient A3 displayed CD8+ T-cell responses to Nef 116. PatientA4 displayed CD8+ T-cell responses to Gag 253, Nef 66, andNef 116 and CD4+ T-cell responses to Gag 17 and Gag 253.Patient A5 displayedCD8+T-cell responses toGag 17, Gag253,

and Nef 66 and CD4+ T-cell responses to Gag 17 and Nef 66.Patient A6 displayed CD8+ T-cell responses to Gag 253 andNef 66 and CD4+ T-cell responses to Gag 253, Nef 66, and Pol325. Interestingly, Nef 66 and Nef 116 were themost frequentCD8+T-cell responses, seen in four of the six patients based onIFN-γ secretion. This assay demonstrated that CD8+ T-cellresponses to the different HIV-1 antigens in the vaccine couldbe elicited by all of the HIV-1 antigen lipopeptides, althougheach patient had unique CD8+ T-cell repertoires and HLAhaplotypes.

3.5. The DC vaccine only activates the peptide specific T-cells

To determine the ability of the DC vaccine to select theHIV-1-specific repertoire, we compared, for two patients (A7and A8), the breadth of the response to the DC vaccine withthat to the single peptides from overlapping HIV Gag and Nefpeptide libraries. As illustrated in Supplementary Fig. 1, ANRSHIV-LIPO-5-loaded DC vaccines were cultured with autolo-gous lymphocytes for 10 days in the presence of IL-2(Supplementary Fig. 1A), or PBMC were cultured with

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Fig. 6. Epitope mapping of antigen-specific T-cells. LIPO-5-loaded DC vaccines from patients A7 and A8 focus the autologous T-cell response on epitopes withinregions of the LIPO-5-antigen peptides. Solid bars along the peptides denote the position where the sequences from the LIPO-5 are located. Red represents Gag 17;Blue represents Gag 253; Purple represents Nef 66; Green represents Nef 116. Autologous T-cell responses were assessed by the amount of IFN-γ (A), IL-13 (B), orIL-10 (C) secreted by cells restimulated with the indicated HIV Gag and Nef peptide libraries. PBMC (light tracings) were primed with the HIV-1-antigen peptidelibraries and DC/LIPO-5 (dark tracings) were autologous lymphocytes primed with the DC vaccine.

35A. Cobb et al. / Journal of Immunological Methods 365 (2011) 27–37

36 A. Cobb et al. / Journal of Immunological Methods 365 (2011) 27–37

individual 15 AA peptides in the presence of IL-2 for 7 days ofculture (Supplementary Fig. 1B). After culture, the cells wererestimulated with the respective peptides for 48 h and IFN-γ,IL-10 and IL-13 levels were measured in the supernatants.

The results from the single peptide analysis indicate abroad repertoire of IFN-γ-producing T-cells in patients A7and A8 (Fig. 6A). Similar results were seen with IL-13(Fig. 6B). Unlike the single peptides, the DC vaccine decreasedthe number of expanded IL-10-secreting T-cells (Fig. 6C).Collectively, these data further demonstrate the efficacy ofLIPO-5-loaded DC vaccines to expand in vitro the memoryCD4+ and CD8+ T-cells in HIV-1 patients undergoing HAART.

4. Discussion

We report here on the pre-clinical development of aprocess for cGMP manufacture of a frozen autologous DCvaccine derived from monocytes isolated from HAART-treated, HIV-1-infected patients. The process consisted ofculturing monocytes with GM–CSF and IFN-α for three days.The resulting DC were then loaded with ANRS HIV-LIPO-5vaccine and activated with LPS. These DC were shown toexpress high levels of MHC Class II and co-stimulatorymolecules. When tested in an in vitro immune potencyassay, the DC vaccines were capable of eliciting allogeneic T-cell proliferation and autologousHIV-1-antigen-specific CD8+

and CD4+ T-cell responses, as measured by an increase in thenumber of IFN-γ-producing T-cells but not IL-10-producing T-cells. Furthermore, the autologous T-cells that were expandedby the DC vaccines were focused on the HIV-1-antigenepitopes expressed with the LIPO-5 lipopeptides.

Previous studies evaluated the use of monocyte-derivedDC in SCID mice and human studies verified the ability ofthese cells to expand HIV-1-specific CD8+ T-cells secretingIFN-γ (Carbonneil et al., 2003; Connolly et al., 2008; Routy etal., 2010) and perforin as well as CD4+ T-cells secreting IFN-γand IL-2 (Lu et al., 2004). Evaluation of vaccinating hu-PBL-SCID mice (SCID mice reconstituted with healthy donorPBMCs) with GM–CSF/IFN-α DC has been shown to inducehigher numbers of HIV-1-specific CD8+ T-cells than thoseelicited by GM–CSF/IL-4 DC. Thus, demonstrating that thetype of DC used for HIV-1-specific vaccination is critical forthe quality and quantity of T-cell responses. This study is alsoevidence of the ability of GM–CSF/IFN-α DC to prime a T-cellresponse in vivo. While this is an important attribute, theintent of the DC vaccine described herein will be used as atherapeutic approach for expanding the T-cell response inHIV-1-infected patients in conjunction with HAART. Vaccina-tion in this population will presumably trigger effector andmemory T-cell responses. As seen in Fig. 6A, IFN-γ productionby HIV-1-antigen-responsive T-cells was seen initially whenperipheral blood lymphocytes were challenged with 15 AAHIV-1-antigen peptides and was further enhanced andfocused with vaccination of the DC vaccine. Furthermore,the DC vaccine dramatically decreased the induction of IL-10-secreting cells, which have been indicative of T regulatorycells (Fig. 6C) (Vignali et al., 2008).

Exploiting the toll-like receptor 2 signaling property of thepalmitoyl-lysylamide lipid tail, it has been previouslyreported that lipopeptides used in vaccine studies are highlyefficient at inducing immune responses, including anti-HIV-1

responses (Martinon et al., 1992; Hosmalin et al., 2001; Zhu etal., 2004). The ANRS HIV-LIPO-5 used in the development ofthe DC vaccine reported in this study has been evaluated innumerous clinical trials, resulting in multiepitopic HIV-1-antigen-specific T-cell responses (Pialoux et al., 2001;Goujard et al., 2007; Launay et al., 2007; Pialoux et al.,2008; Salmon-Ceron et al., 2010). Based on the Los Alamosdatabase and ANRS data, each of the HIV-1 antigen peptidespresent in LIPO-5 is recognized by numerous HLA haplotypes(Supplementary Table 3) and has been documented to be partof conserved regions of HIV-1 antigens (Salmon-Ceron et al.,2010). This is an important criterion for the development of avaccine that can treat as many patients as possible with littleimmunological restrictions due to HLA haplotype differences.Furthermore, it should be noted that the frequency of the T-cell precursors is variable per patient, which will result indifferent epitopes presented (as seen in Fig. 5B).

Combining the two strategies, GM–CSF/IFN-α DC andLIPO-5, has become an attractive union for inducing a broadpotent effector immune response. This is the first combina-tion strategy for inducing effector immune responses tobe tested in humans. Thus, a Phase I/II clinical trial has beeninitiated and is nearing completion (clinicaltrials.gov identi-fier: NCT00796770) for therapeutic vaccination in chronicallyHIV-1-infected patients on HAART.

Supplementary materials related to this article can befound online at doi:10.1016/j.jim.2010.11.002.

Acknowledgements

We thank the volunteers for participation in this study.We thank the medical staff from North Texas InfectiousDiseases Consultants. We thank Dr. M. Ramsay and C.Samuelsen for continuous support. A.K.P. holds the RamsayChair for Cancer Immunology. We thank Eynav Klechevskyfor technical discussions. The development of this DC vaccinewas supported by the French National Agency for AIDSResearch (ANRS) Paris, France and by grants from BHCSFoundation. Author contribution: A.C. designed and per-formed the research, analyzed results, made the figures andwrote the manuscript; H.M., M.M., R.R., S.B., and J.P.Fcontributed technical assistance, D.B. contributed statisticalanalysis, A.C.H. contributed regulatory and administrativeassistance, B.K. and L.S. provided HIV-1 samples for research,Y.L., A.K.P., L.K.R. and J.B designed the research and wrote themanuscript.

References

Alvarez, D., Vollmann, E.H., von Andrian, U.H., 2008. Mechanisms andconsequences of dendritic cell migration. Immunity 29, 325.

Appay, V., Nixon, D.F., Donahoe, S.M., Gillespie, G.M., Dong, T., King, A., Ogg, G.S.,Spiegel, H.M., Conlon, C., Spina, C.A., Havlir, D.V., Richman, D.D., Waters, A.,Easterbrook, P.,McMichael, A.J., Rowland-Jones, S.L., 2000.HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolyticfunction. J. Exp. Med. 192, 63.

Banchereau, J., Steinman, R.M., 1998. Dendritic cells and the control ofimmunity. Nature 392, 245.

Banchereau, J., Ueno, H., Dhodapkar, M., Connolly, J., Finholt, J.P., Klechevsky,E., Blanck, J.P., Johnston, D.A., Palucka, A.K., Fay, J., 2005. Immune andclinical outcomes in patients with stage IV melanoma vaccinated withpeptide-pulsed dendritic cells derived from CD34(+) progenitors andactivated with type I interferon. J. Immunother. 28, 505.

37A. Cobb et al. / Journal of Immunological Methods 365 (2011) 27–37

Bonaldo, M.C., Martins, M.A., Rudersdorf, R., Mudd, P.A., Sacha, J.B.,Piaskowski, S.M., Neves, P.C.C., de Santana, M.G.V., Vojnov, L., Capuano,S., Rakasz, E.G., Wilson, N.A., Fulkerson, J., Sadoff, J.C., Watkins, D.I., Galler,R., 2010. Recombinant yellow fever vaccine virus 17D expressing simianimmunodeficiency virus SIVmac239 Gag induces SIV-specific CD8(+) T-cell responses in Rhesus Macaques. J. Virol. 84, 3699.

Borrow, P., Lewicki, H., Hanh, B.H., Shaw, G.M., Oldstone, M.B., 1994. Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control ofviremia in primary human immunodeficiency virus type 1 infection. J.Virol. 68, 6103.

Cao, Y., Qin, L., Zhang, L., Safrit, J., Ho, D.D., 1995. Virologic and immunologiccharacterization of long-term survivors of human immunodeficiencyvirus type 1 infection. N. Engl. J. Med. 332, 201.

Carbonneil, C., Aouba, A., Burgard, M., Cardinaud, S., Rouzioux, C., Langlade-Demoyen, P., Weiss, L., 2003. Dendritic cells generated in the presence ofgranulocyte–macrophage colony-stimulating factor and IFN-alpha arepotent inducers of HIV-specific CD8 T cells. AIDS 17, 1731.

Carcelain, G., Tubiana, R., Samri, A., Calvez, V., Delaugerre, C., Agut, H.,Katlama, C., Autran, B., 2001. Transient mobilization of humanimmunodeficiency virus (HIV)-specific CD4 T-helper cells fails to controlvirus rebounds during intermittent antiretroviral therapy in chronic HIVtype 1 infection. J. Virol. 75, 234.

Champagne, P., Ogg, G.S., King, A.S., Knabenhans, C., Ellefsen, K., Nobile, M.,Appay, V., Rizzardi, G.P., Fleury, S., Lipp, M., Forster, R., Rowland-Jones, S.,Sekaly, R.P., McMichael, A.J., Pantaleo, G., 2001. Skewed maturation ofmemory HIV-specific CD8 T lymphocytes. Nature 410, 106.

Connolly, N.C., Whiteside, T.L., Wilson, C., Kondragunta, V., Rinaldo, C.R.,Riddler, S.A., 2008. Therapeutic immunization with human immunode-ficiency virus type 1 (HIV-1) peptide-loaded dendritic cells is safe andinduces immunogenicity in HIV-1-Infected individuals. Clin. VaccineImmunol. 15, 284.

Deeks, S.G., Walker, B.D., 2007. Human immunodeficiency virus controllers:mechanisms of durable virus control in the absence of antiretroviraltherapy. Immunity 27, 406.

Durier, C., Launay, O., Meiffredy, V., Saidi, Y., Salmon, D., Levy, Y., Guillet, J.G.,Pialoux, G., Aboulker, J.P., 2006. Clinical safety of HIV lipopeptides used asvaccines in healthy volunteers and HIV-infected adults. AIDS 20, 1039.

Goepfert, P.A., Bansal, A., Edwards, B.H., Ritter Jr., G.D., Tellez, I., McPherson, S.A.,Sabbaj, S., Mulligan, M.J., 2000. A significant number of human immuno-deficiency virus epitope-specific cytotoxic T lymphocytes detected bytetramer binding do not produce gamma interferon. J. Virol. 74, 10249.

Goujard, C., Marcellin, F., Hendel-Chavez, H., Burgard, M., Meiffredy, V.,Venet, A., Rouzioux, C., Taoufik, Y., El Habib, R., Beumont-Mauviel, M.,Aboulker, J.P., Levy, Y., Delfraissy, J.F., Grp, P.-A.S., 2007. Interruption ofantiretroviral therapy initiated during primary HIV-1 infection: Impactof a therapeutic vaccination strategy combined with interleukin (IL)-2compared with IL-2 alone in the ANRS 095 randomized study. AIDS Res.Hum. Retroviruses 23, 1105.

Hosmalin, A., Andrieu, M., Loing, E., Desoutter, J.F., Hanau, D., Gras-Masse, H.,Dautry-Varsat, A., Guillet, J.G., 2001. Lipopeptide presentation pathwayin dendritic cells. Immunol. Lett. 79, 97.

Kaufmann, G.R., Zaunders, J.J., Cunningham, P., Kelleher, A.D., Grey, P., Smith,D., Carr, A., Cooper, D.A., 2000. Rapid restoration of CD4 T cell subsets insubjects receiving antiretroviral therapy during primary HIV-1 infection.AIDS 14, 2643.

Koup, R.A., Safrit, J.T., Yunzhen, C., Andrews, C.A., McLeod, G., Borkowsky, W.,Farthing, C., Ho, D.D., 1994. Temporal association of cellular immuneresponses with the initial control of viremia in primary humanimmunodeficiency virus type 1 syndrome. J. Virol. 68, 4650.

Launay, O., Durier, C., Desaint, C., Silbermann, B., Jackson, A., Pialoux, G., Bonnet,B., Poizot-Martin, I., Gonzalez-Canali, G., Cuzin, L., Figuereido, S., Surenaud,M., Ben Hamouda, N., Gahery, H., Choppin, J., Salmon, D., Guerin, C., Villada,I.B., Guillet, J.G., Grp, A.V.S., 2007. Cellular immune responses induced withdose-sparing intradermal administration of HIV vaccine to HIV-uninfectedvolunteers in the ANRS VAC16 trial. PLoS ONE 2 -.

Levy, Y., Gahery-Segard, H., Durier, C., Lascaux, A.S., Goujard, C., Meiffredy, V.,Rouzioux, C., El Habib, R., Beumont-Mauviel, M., Guillet, J.G., Delfraissy, J.F.,

Aboulker, J.P., Grp, A.S., 2005. Immunological and virological efficacy of atherapeutic immunization combinedwith interleukin-2 in chronicallyHIV-1 infected patients. AIDS 19, 279.

Liu, J.Y.,O'Brien, K.L., Lynch,D.M., Simmons,N.L., LaPorte,A., Riggs, A.M., Abbink,P., Coffey, R.T., Grandpre, L.E., Seaman, M.S., Landucci, G., Forthal, D.N.,Montefiori, D.C., Carville, A., Mansfield, K.G., Havenga, M.J., Pau, M.G.,Goudsmit, J., Barouch, D.H., 2009. Immune control of an SIV challenge by aT-cell-based vaccine in rhesus monkeys. Nature 457, 87.

Lu, W., Arraes, L.C., Ferreira, W.T., Andrieu, J.M., 2004. Therapeutic dendritic-cell vaccine for chronic HIV-1 infection. Nat. Med. 10, 1359.

Martinon, F., Grasmasse, H., Boutillon, C., Chirat, F., Deprez, B., Guillet, J.G.,Gomard, E., Tartar, A., Levy, J.P., 1992. Immunization of mice withlipopeptides bypasses the prerequisite for adjuvant—immune-responseof Balb/C mice to human-immunodeficiency-virus envelope glycopro-tein. J. Immunol. 149, 3416.

McMichael, A.J., Borrow, P., Tomaras, G.D., Goonetilleke, N., Haynes, B.F.,2010. The immune response during acute HIV-1 infection: clues forvaccine development. Nat. Rev. Immunol. 10, 11.

Melief, C.J.M., 2008. Cancer immunotherapy by dendritic cells. Immunity 29,372.

Palucka, A.K., Ueno, H., Fay, J.W., Banchereau, J., 2007. Taming cancer byinducing immunity via dendritic cells. Immunol. Rev. 220, 129.

Pialoux, G., Gahery-Segard, H., Sermet, S., Poncelet, H., Fournier, S., Gerard, L.,Tartar, A., Gras-Masse, H., Levy, J.P., Guillet, J.G., Team, A.V.S., 2001.Lipopeptides induce cell-mediated anti-HIV immune responses inseronegative volunteers. AIDS 15, 1239.

Pialoux, G., Quercia, R.P., Gahery, H., Daniel, N., Slama, L., Girard, P.M.,Bonnard, P., Rozenbaum, W., Schneider, V., Salmon, D., Guillet, J.G., 2008.Immunological responses and long-term treatment interruption afterhuman immunodeficiency virus type 1 (HIV-1) lipopeptide immuniza-tion of HIV-1-infected patients: the LIPTHERA study. Clin. VaccineImmunol. 15, 562.

Rosenberg, E.S., Billingsley, J.M., Caliendo, A.M., Boswell, S.L., Sax, P., Kalams,S.A., Walker, B.D., 1997. Vigorous HIV-1-specific CD4+ T cell responsesassociated with control of viremia. Science 278, 1447.

Routy, J.P., Boulassel, M.R., Yassine-Diab, B., Nicolette, C., Healey, D., Jain, R.,Landry, C., Yegorov, O., Tcherepanova, I., Monesmith, T., Finke, L., Sekaly,R.P., 2010. Immunologic activity and safety of autologous HIV RNA-electroporated dendritic cells in HIV-1 infected patients receivingantiretroviral therapy. Clin. Immunol. 134, 140.

Sabado, R.L., Bhardwaj, N., 2010. Directing dendritic cell immunotherapytowards successful cancer treatment. Immunotherapy-Uk 2, 37.

Salmon-Ceron, D., Durier, C., Desaint, C., Cuzin, L., Surenaud, M., BenHamouda, N., Lelievre, J.D., Bonnet, B., Pialoux, G., Poizot-Martin, I.,Aboulker, J.P., Levy, Y., Launay, O., Grp, A.V.T., 2010. Immunogenicity andsafety of an HIV-1 lipopeptide vaccine in healthy adults: a phase 2placebo-controlled ANRS trial. AIDS 24, 2211.

Steinman, R.M., Banchereau, J., 2007. Taking dendritic cells into medicine.Nature 449, 419.

Timmerman, J.M., Levy, R., 2000. Linkage of foreign carrier protein to a self-tumor antigen enhances the immunogenicity of a pulsed dendritic cellvaccine. J. Immunol. 164, 4797.

Vignali, D.A.A., Collison, L.W., Workman, C.J., 2008. How regulatory T cellswork. Nat. Rev. Immunol. 8, 523.

Virgin, H.W., Walker, B.D., 2010. Immunology and the elusive AIDS vaccine.Nature 464, 224.

Yu, C.I., Gallegos, M., Marches, F., Zurawski, G., Ramilo, O., Garcia-Sastre, A.,Banchereau, J., Palucka, A.K., 2008. Broad influenza-specific CD8(+) T-cell responses in humanized mice vaccinated with influenza virusvaccines. Blood 112, 3671.

Zhu, X.M., Ramos, T.V., Gras-Masse, H., Kaplan, B.E., BenMohamed, L., 2004.Lipopeptide epitopes extended by an N-epsilon-palmitoyl-lysine moietyincrease uptake and maturation of dendritic cells through a toll-likereceptor-2 pathway and trigger a Th1-dependent protective immunity.Eur. J. Immunol. 34, 3102.