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Vaccine 27 (2009) 6939–6949

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

iposome-encapsulated HIV-1 Gag p24 containing lipid A induces effector CD4+-cells, memory CD8+ T-cells, and pro-inflammatory cytokines�

icholas J. Steers, Kristina K. Peachman, Sasha McClain, Carl R. Alving, Mangala Rao ∗

ivision of Retrovirology, USMHRP, Walter Reed Army Institute of Research, 1600 E. Gude Drive, Rockville, MD 20850, USA

r t i c l e i n f o

rticle history:eceived 7 July 2009eceived in revised form 26 August 2009ccepted 27 August 2009vailable online 11 September 2009

eywords:

a b s t r a c t

Liposomal lipid A is an effective adjuvant for the delivery of antigens and for the induction of both cellularand humoral immunity. In this study, we demonstrate that following the third immunization with HIV-1Gag p24 encapsulated in liposomes containing lipid A [L(p24 + LA)], central memory CD8+ T-cells werelocalized in the spleen and lymph nodes of mice while effector memory CD8+ T-cells and effector CD4+T-cells were found in the PBMC. Effector CD4+ T-cells were also detected in the spleen and lymph nodes.The predominant cytokine secreted from splenic lymphocytes and lymph nodes was IFN-�. In contrast,

posomal lipid Aemory CD8+ T-cells

IL-6 and IL-10 were the major cytokines produced from PBMC. The peptide stimulation indicated that thecytokine responses observed were T-cell specific. The results demonstrate the importance of the adjuvant

indue pro

ffector CD4+ T-cells liposomal lipid A for thecytokines with a Th-1 typ

. Introduction

Inducing an efficient and protective immune response againstathogens is the goal in any vaccination strategy. In order to achievehis, a candidate vaccine should activate antigen-presenting cellsor the efficient priming of the adaptive arm of the host’s immuneesponse. Antigens need to be delivered to antigen-presentingells for processing and presentation by MHC class I and classI molecules to the appropriate CD8+ and CD4+ T-cells, respec-ively. Immunization with a protein or peptide antigen alone inhe absence of an adjuvant fails to effectively prime the immuneesponse [1–3]. One specific adjuvant that we have extensively usedn several studies is liposomal lipid A [L(LA)] [4–7]. Lipid A is thendotoxic moiety of gram-negative bacterial lipopolysaccharide.ntigens incorporated into liposomes containing lipid A have beensed in animal studies and human clinical trials for the induction ofrobust cellular, humoral, and mucosal immune response [5–15].

We have previously demonstrated that L(LA) is a potent inducerf antigen-presenting cells [7]. In vitro studies have shown that

ntigens encapsulated in liposomes are readily taken up byoth macrophages and dendritic cells and that the antigens arefficiently processed. The resulting antigenic peptides are pre-ented by MHC class I and class II molecules [16–18]. L(LA) also

The views expressed in this article are those of the authors and do not reflecte official policy of the Department of the Army, the Department of Defense, or the.S. Government.∗ Corresponding author. Tel.: +1 301 251 5019; fax: +1 301 424 3120.

E-mail address: mrao@hivresearch.org (M. Rao).

264-410X/$ – see front matter. Published by Elsevier Ltd.oi:10.1016/j.vaccine.2009.08.105

ction of HIV-1 Gag p24 -specific CD8+ T-cells, effector CD4+ T-cells, andfile after immunization with L(p24 + LA).

Published by Elsevier Ltd.

causes the phenotypic maturation of dendritic cells, activation ofmacrophages, induction of pro-inflammatory cytokines, and themodulation of immunoproteasomes [19]. L(LA) thus appears to actas an intracellular adjuvant by upregulating the antigen processingmachinery, which results in efficient antigen presentation. Otherstudies using liposomal antigens and adjuvants such as CAF01 [20]and CpG [21] have demonstrated that the use of adjuvants alongwith antigens are required for the induction of activated CD4+ andCD8+ T-cells including memory cells.

A potential vaccine should have the ability to induce and main-tain either antigen-specific effector and/or central memory T-cells,which can be easily expanded following re-exposure to the antigensduring an infection. Memory T-cells not only reside in the spleenand lymph nodes but are also found in the peripheral immuneorgans such as the liver, kidney, and lungs [22–24]. Following eitherinfection or vaccination, CD4+ and CD8+ T-cells display the charac-teristic memory or effector markers. The effector/memory cells arelocated around the area of infection or at the site of vaccination. Tra-ditionally, the induction of effector/central memory cells has beenexamined in the spleen and lymph nodes and not in peripheralimmune organs where the disease occurs. However, in influenza[25], TB [26], and malaria [22], the induction of memory effectorcells has also been characterized in specific peripheral immuneorgans such as the lungs and liver.

Memory T-cells express specific cell surface molecules, which

determine the effector status and the type of memory cell. Inmice, the best-characterized markers are CD44, CD45Rb, CD62Land CD122. CD62L, also known as l-selectin or lymph node hom-ing molecule, is expressed at low levels on effector CD4+ T-cellsand effector memory CD8+ T-cells [27]. Both CD44 and CD45Rb

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ell surface molecules are associated with effector CD4+ and CD8+emory T-cells. All effector memory cells express high levels of

D44 [27,28]. However, central memory CD8+ T-cells express highevels of CD45Rb while effector memory CD8+ T-cells express lowevels of CD45Rb [29]. CD122, the IL-2/IL-15 receptor, expressedn the surface of central memory CD8+ T-cells requires IL-15 forheir maintenance and survival. On effector memory CD8+ T-cells,D122 is expressed in low levels [27,30].

Antigen-specific memory T-cells secrete cytokines, which causehe rapid clearance of the invading pathogen, thus preventing hostnfection. The effector memory CD8+ T-cells are generally short-ived and die in the absence of constant exposure to the antigen31,32]. However, a small percentage of these antigen-specific CD8+-cells persist [33] as central memory CD8+ T-cells and are main-ained within the lymph nodes and spleens. Upon re-infection,hese cells are capable of rapid proliferation and develop into effec-or memory CD8+ T-cells, which are capable of rapid removal of theathogen.

The induction of effector CD4+ T-cells occurs in the same mannernd with similar dynamics as is seen with the induction of effec-or memory CD8+ T-cells. Following activation, naive CD4+ T-cellsifferentiate into effector cells with various functions, includ-

ng providing help for the induction of antigen-specific memoryD8+ T-cells and the secretion of cytokines. Effector CD4+ T-cellsre capable of secreting both Th1 (mainly IFN-�) and Th2 typeytokines (mainly IL-4 and IL-5) [34,35]. Like the memory CD8+-cells, the effector CD4+ T-cells also have a rapid recall responseollowing re-exposure to the antigen [36,37] and require the pres-nce of the antigen for its maintenance and survival.

It is well known that in HIV-infected individuals the CD4+ T-cellsre dysfunctional. It has also been demonstrated that one of the ear-iest events after infection with HIV or SIV is a dramatic destructionf CD4+ memory T-cells [38]. Studies in a macaque model indicatehat vaccination preserves CD4+ central memory T-cells and affordsetter protection against SIV challenge [39,40]. Therefore, vaccinesapable of generating and maintaining HIV-specific T-cell effectoremory cells might decrease the incidence of HIV acquisition after

xposure.In the present study, Gag p24 was chosen as the antigen because

ag is frequently used as one of the components of candidate HIV-vaccines. Gag is fairly conserved and T cell responses to Gag

ave been shown to directly correlate with viremia [41,42]. Wenvestigated the generation of central/effector memory CD8+ T-ells, effector CD4+ T-cells and the induction of pro-inflammatoryytokines from the spleens, lymph nodes, and peripheral blood ofice immunized by the intramuscular route with HIV-1 Gag p24

ncapsulated in liposomes with lipid A [L(p24 + LA)]. The resultsere compared with mice immunized with empty liposomes with

L(LA)] and without lipid A [(L)] and p24 or p24 encapsulatedn liposomes [L(p24)]. The expansion of effector CD4+ T-cells,enoted by CD44hi CD62Llow was observed in the spleen, lymphodes, and peripheral blood, following the third immunizationith L(p24 + LA). Central memory CD8+ T-cells (CD44hi, CD45RBhi,D62Lhi) were observed following immunization with L(p24 + LA).ffector memory CD8+ T-cells (CD44hi, CD45RBlow, CD62Llow) werenduced only in the peripheral blood following immunization with(p24 + LA). Lymphocytes obtained from mice immunized with(p24 + LA) and cultured in vitro with either p24 or the p24-CTL-eptide induced a predominantly Th1 type cytokine response.

. Materials and methods

.1. Animals

Female C57BL/6 mice (6–8 weeks) were purchased from theackson labs (Bar Harbor, MA) and used for immunization studies.

7 (2009) 6939–6949

The study was conducted in compliance with the animal welfare actand adhered to the principles in the guide for care and use of lab-oratory animals. The investigators used facilities accredited by theAssociation for Assessment and Accreditation of Laboratory Ani-mal Care International. The Walter Reed Army Institute of ResearchAnimal Care and Use Committee approved all animal experiments.

2.2. Antigen

For immunization of mice, Baculovirus-expressed Gag p24 HIV-1111B was purchased from Immunodiagnostics, Inc. (Woburn, MA).Purified E. coli produced HIV-1 Gag p24 [43] was obtained from theCatholic University of America and used for in vitro stimulation. Theendotoxin level determined by Limulus Amebocyte Lysate assay(LAL, Associates of Cape Cod Incorporated, E. Falmouth, MA) was≤50 pg/mL.

2.3. Liposome preparation

Detailed procedures for the preparation of liposomes weredescribed previously [19,44]. Briefly, multilamellar liposomescomposed of dimyristoyl phosphatidylcholine, dimyristoyl phos-phatidylglycerol and cholesterol (Avanti polar lipids, Alabaster, AL)in molar ratios of 1.8:0.2:1.5 with or without 1 mg/mL lipid A (ListBiological Labs., Campbell, CA) were prepared by dispersion oflyophilized mixtures of lipids at a phospholipid concentration of100 mM in Dulbecco’s PBS either lacking or containing HIV-1 Gagp24. Liposomes were washed twice in sterile saline to remove theunencapsulated antigen, resuspended in 1 mL of sterile saline andstored at 4 ◦C until used. The amount of antigen encapsulated inliposomes was determined by a modified Lowry procedure [44,45].

2.4. Immunization of mice

C57BL/6 mice (three mice per group) were immunized by theintramuscular route on days 0, 21, and 42 with 50 �L of the follow-ing immunogens: unencapsulated liposomes (L), liposomal lipid A[L(LA)], HIV-1 Gag p24 encapsulated in liposomes lacking lipid A[L(p24)] (10.4 �g of p24), HIV-1 Gag p24 encapsulated in liposomescontaining lipid A [L(p24 + LA)] (15.2 �g of p24) or p24 (25 �g).Mice were euthanized 3 days following each immunization andthe spleens, lymph nodes, and peripheral blood were harvested foranalysis by flow cytometry and for the determination of cytokinessecreted after in vitro stimulation with antigens. In each case, age-matched naive mice were used as the controls.

2.5. Flow cytometry analysis

Single cell preparations were prepared from spleens and lymphnodes of naive and immunized mice. Whole blood was collectedfrom individual mice at each of the time points. In all cases, RBCswere lysed using ACK buffer. Cells (1 × 106) were resupended inflow buffer (PBS containing 1% BSA) and pre-incubated with anti-mouse CD32/16 antibody (Fc Block, 1 �g/1 × 106 cells) for 10 minat 4 ◦C before staining the cells for surface markers. Cells werestained with CD4, CD44, and CD62L for CD4+ T-cells and with CD8,CD44, CD45Rb, and CD62L for CD8+ T-cells, respectively. Cells werewashed twice in ice-cold flow buffer and fixed in 2% formaldehydein PBS, before analysis on a FACScalibur (BD Biosciences) using CellQuest (BD Biosciences) and Flow Jo (TreeStar, Ashland, OR) soft-ware. CD4+ T-cells were gated from the lymphocyte population

and then analyzed for the expression of CD44 and CD62L. EffectorCD4+ T-cells were characterized as CD4+, CD44hi, and CD62Llow.Naive CD4+ T-cells were characterized as CD4+, CD44inter/low, andCD62Lhi. CD8+ memory T-cells were gated from the lymphocytepopulation and analyzed for the expression of CD44, CD45Rb, and

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D62L. CD8+ central memory T-cells were characterized as CD8+,D44hi, CD45Rbhi, and CD62Lhi and CD8+ effector memory T-cellss CD8+, CD44hi, CD45Rblow, and CD62Llow.

.6. Lymphocyte cultures

All manipulations of spleens, lymph nodes, and peripheral bloodere performed under aseptic conditions. Lymph node and periph-

ral blood samples were pooled, whereas the spleen cells fromndividual mice were cultured separately. Cells (5 × 106/mL) wereesuspended in complete RPMI-1640 (Invitrogen) containing l-lutamine, 10% FCS and 1% pen/strep and plated in 24-well sterileissue culture plates. Cells were cultured in media alone, con-anavalin A (10 �g/mL) as a positive control, E. coli expressed p245 �g/mL), or with the p24-peptide HQAISPRTLNAWVKV (1 �g/mL)hat was synthesized in-house. The reported MHC class I H-2b epi-ope is shown in italics [46]. After 48 h, the culture supernatantsere collected, clarified by centrifugation, and stored at −80 ◦Cntil analyzed.

.7. Cytokine determination

IL-2 and IL-4 in the supernatants were determined in duplicatessing the appropriate cytokine capture ELISA kits and assayed aser the manufacturer’s instructions (OptEIA, BD Biosciences). Sam-les were read using a SpectraMax 250 plate reader (Molecularevices, Palo Alto, CA). The lower limit of detection in the culture

upernatants for IL-2 and IL-4 was 3.1 and 7.8 pg/mL, respectively.L-6, IL-10, IL-12p70, IFN-�, TNF-�, and MCP-1 were measuredsing mouse Cytometric Bead Array inflammation kit (BD Bio-ciences). For each cytokine, 300 data points were collected andnalyzed using a FACScalibur. The cytokine concentration wasalculated from the mean fluorescent intensity as per the manu-acturer’s instructions. The lower limit of detection for each of theytokines in the culture supernatants was 20 pg/mL.

. Results

We have previously demonstrated that immunization of miceith antigens encapsulated in liposomes containing lipid A causes

he activation of antigen-presenting cells and induces a potentumoral and cellular immune response including CTL responses5–19]. In the present study, we examined the induction of mem-ry CD8+ and effector CD4+ T-cells in the spleen, lymph node, anderipheral blood mice 3 days post-immunization with either p24lone, L, L(LA), L(p24) or L(p24 + LA). Each data point was measuredn duplicate for lymph node and peripheral blood samples and inriplicate for spleen cells. The experiment was repeated twice withaive and L(p24 + LA) immunized mice. Age-matched naive miceere used at each time point and served as one of the controls. A

epresentative experiment is shown in Figs. 1–3.

.1. Induction of memory CD8+ T-cells and effector CD4+ T-cellsn the spleen and lymph nodes after immunization with(p24 + LA)

Single cell suspensions of the spleen and lymph nodes wereated for either CD8+ or CD4+ T-cells. The CD8+ T-cells were thennalyzed for the surface expression of CD44, CD45Rb, and CD62L.entral memory CD8+ T-cells were characterized as CD8+, CD44hi,D45Rbhi, and CD62Lhi and effector memory CD8+ T-cells as CD8+,

D44hi, CD45Rblow, and CD62Llow. The CD4+ T-cells were thennalyzed for the surface expression of CD44 and CD62L. EffectorD4+ T-cells were characterized as CD4+, CD44hi, and CD62Llow.aive CD4+ T-cells were characterized as CD4+, CD44inter/low, andD62Lhi.

7 (2009) 6939–6949 6941

Flow cytometry data of spleens (Fig. 1B and C and Table 1) frommice immunized with L(p24 + LA) (Fig. 1A) and lymph nodes (Fig. 2and Table 2) indicated that there was a significant induction [13.7%for immune spleen cells vs. 11% for naive (p < 0.0005); 14.2% forimmune lymph node cells vs. 11.3% for naive (p = 0.04)] of centralmemory (CM) CD8+ T-cells (CD44hi, CD45RBhi, CD62Lhi) in bothimmune organs (Figs. 1B and 2A) following the third immuniza-tion with L(p24 + LA) in comparison to age-matched naive controlmice. The data also showed the appearance of effector memory(EM) CD8+ T-cell population (CD44hi, CD45RBlow, CD62Llow-inter)in the spleen [Fig. 1B; 2.22% for immune spleen cells vs. 0.54% fornaive (p < 0.0001)] but not in the lymph node cells (Fig. 2A). A sig-nificant induction of effector CD4+ T-cells (CD44hi, CD62Llow) waspredominantly seen in the spleens [Fig. 1C; 17.5% for immune cellsvs. 6.65% for naive (p < 0.0007)] and lymph nodes [Fig. 2B; 10.1% forimmune cells vs. 5.71% for naive (p < 0.0002)] of mice immunizedwith L(p24 + LA).

Immunization with p24, L, L(LA) or L(p24) showed no induc-tion of central or effector memory CD8+ T-cells in the spleens(Table 1) and lymph nodes (Table 2) and that the majority ofthe cells remained in the naive status (CD44low) in comparisonto age-matched naive control mice. Following the third immu-nization with L(p24 + LA), effector CD4+ T-cells (CD44hi, CD62Llow)were induced in the spleens (15.36% for immune vs. 6.1% for naïve,Table 1) and lymph nodes (12.9% for immune vs. 6.15% for naive,Table 2). The percentage of effector CD4+ T-cells found in the spleen(Table 1) and lymph nodes (Table 2) following immunization withp24 alone or with L(p24) were similar to that observed with thecontrol immunizations using L or L(LA). The above results demon-strate that for the induction of memory CD8+ and effector CD4+T-cells, the adjuvant lipid A was required along with p24.

3.2. Induction of effector memory CD8+ T-cells and effector CD4+T-cells in peripheral blood after immunization with L(p24 + LA)

Effector memory CD8+ T-cells (CD44hi, CD45RBlow, CD62Llow)were observed in the peripheral blood following the third immu-nization (Fig. 3A) and accounted for 10.6% of the CD8+ T-cellscompared to 0% in age-matched control mice (p < 0.0001). Theseeffector memory CD8+ T-cells were only seen after the thirdimmunization with L(p24 + LA) and not following immunizationswith either L, L(LA), L(p24) or p24 alone (Table 3). There was noincrease in the percentage of central memory CD8+ T-cells (CD44hi,CD45RBhi, CD62Lhi) following the third immunization with p24,L(p24) and L(p24 + LA) when compared to peripheral blood lym-phocytes from age-matched control mice and mice immunizedwith L and L(LA) (Fig. 3A and Table 3).

Effector CD4+ T-cells were observed after the third immuniza-tion with either p24 (14.8%), L(p24) (17.6%) or L(p24 + LA) (28.01%)compared to age-matched control mice (9.08%, Table 3). However,the greatest induction of effector CD4+ T-cells was observed in miceimmunized with L(p24 + LA) (Fig. 3B and Table 3). Immunizationwith L(p24 + LA) significantly increased the percentage of effectorCD4+ T-cells from 9.9% (naive) to 26.5% [Fig. 3B; p < 0.0001] and9.08% (naive) to 28.01% (Table 3), respectively with a concomitantdecrease (75.2% in naive mice vs. 58.9% in immune mice, Fig. 3B) inphenotypically classified non-effector CD4+ T-cells.

These data indicate that immunization with L(p24 + LA) causesthe induction of both central and effector memory CD8+ T-cells.Effector memory CD8+ T-cells were predominantly identified in theperipheral blood and CD8+ central memory T-cells were identified

in the spleen and lymph node. Effector CD4+ T-cells were not local-ized to any specific immune organ but instead were identified in thespleen, lymph node, and peripheral blood. These data also showsthe importance of the adjuvant liposomal lipid A for the inductionof memory and effector T-cells.

6942 N.J. Steers et al. / Vaccine 27 (2009) 6939–6949

Fig. 1. Central memory CD8+ T-cells and effector CD4+ T-cells are increased in the spleens of mice immunized with L(p24 + LA). (A) C57BL/6 mice were immunized withL(p24 + LA) on days 0, 21, and 42. Three days after each immunization, individual spleens were collected from three immunized and three age-matched naive mice. Single cellsuspensions were stained for memory CD8+ and effector CD4+ T-cells. Cells were analyzed by four-color flow cytometry. (B) Central memory CD8+ T-cells were characterizedas CD44hi, CD45Rbhi, and CD62Lhi while effector memory CD8+ T-cells were characterized as CD44hi, CD45Rblow and CD62Llow. A slight induction of both central (13.7%) andeffector (2.22%) memory CD8+ T-cells was observed in the spleen post-third immunization compared to age-matched naive mice (11% and 0.54%, respectively). (C) CD4+T-cells were stained with CD44 and CD62L. Effector CD4+ T-cells (CD44hi and CD62Llow) and naive CD4+ T-cells (CD44low and CD62Lhi) were characterized. There was a stronginduction of effector CD4+ T-cells (17.3% for immune mice vs. 8.9% for naive mice) following the second immunization that was also seen after the third immunization (17.5%for immune mice vs. 6.65% for naive mice) compared to age-matched naive mice. The data shown is a representative of one individual mouse of a group of three mice. Theexperiment was repeated twice and the average of six mice from two separate experiments is shown in Table 1.

N.J. Steers et al. / Vaccine 27 (2009) 6939–6949 6943

Fig. 2. Central memory CD8+ T-cells and effector CD4+ T-cells are increased in the lymph nodes of mice immunized with L(p24 + LA). See Fig. 1 caption for details forthe determination of central and effector memory CD8+ T-cells and effector CD4+ T-cells. The results represent pooled lymphocytes from the lymph nodes of three miceimmunized with L(p24 + LA) and three age-matched naive mice. (A) A slight induction of central memory CD8+ T-cells was observed in the immunized mice following the thirdimmunization compared to the age-matched control mice (14.2% for immune mice vs. 11.3% for naive mice). (B) An increase in the effector CD4+ T-cells was detected in thelymph nodes following the third immunization compared to age-matched control mice (10.1% for immune mice vs. 5.71% for naive mice). The data shown is a representativeexperiment of pooled lymph nodes from three mice from two separate experiments.

6944 N.J. Steers et al. / Vaccine 27 (2009) 6939–6949

Fig. 3. Effector memory CD8+ T-cells and effector CD4+ T-cells are increased in the peripheral blood mononuclear cells of mice immunized with L(p24 + LA). See Fig. 1 captionfor details. The results represent pooled lymphocytes from the peripheral blood of immunized and age-matched naive mice. (A) Following the third immunization, effectormemory CD8+ T-cells (CD44hi, CD45Rblow and CD62Llow) were induced compared to age-matched control mice (10.6% for immune mice vs. 0% for naive mice). (B) EffectorCD4+ T-cells were observed in the PBMC only after the third immunization compared to the age-matched control mice (26.5% for immune mice vs. 9.9% for naive mice). Thedata shown is a representative experiment of pooled PBMC from three mice from two separate experiments.

N.J. Steers et al. / Vaccine 27 (2009) 6939–6949 6945

Table 1Characterization of CD8+ and CD4+ T-cells in the spleens of immunized mice 3 days following the third immunizationa.

Immunogen % CM CD8+ T-cells CD44hi, CD45Rbhi, CD62Lhi % EM CD8+ T-cells CD44hi, CD45Rblow, CD62Llow % Eff CD4+ T-cells CD44hi, CD62Llow

Naive 9.04 ± 1.4 0 6.1 ± 0.98L 9.12 ± 1.4 0 8.16 ± 2.4L(LA) 8.91 ± 1.3 0 9.45 ± 1.6p24 10.8 ± 0.6 0 7.39 ± 2.7L(p24) 9.43 ± 1.6 0 9.42 ± 2.4L(p24 + LA) 12.7 ± 0.9* 2.34 ± 0.45* 15.36 ± 2.4*

a Single cell suspensions were prepared from the spleens of mice 3 days after the second boost with either L, L(LA), p24, L(p24) or L(p24 + LA) and compared with age-matched control mice. The cells were stained with CD8, CD44, CD45Rb and CD62L or with CD4, CD44 and CD62L and then analyzed by flow cytometry. Central memory (CM)CD8+ T-cells were characterized as CD44hi, CD45Rbhi and CD62Lhi. Effector memory (EM) CD8+ T-cells were characterized as CD44hi, CD45Rblow and CD62Llow and effectorCD4+ T-cells as CD4+ CD44hi, and CD62Llow. The data represents the average of two separate experiments (three individual spleens for each experiment) from naive andL(p24 + LA) immunized mice and one experiment (three individual spleens) from mice immunized with either L, (LA), p24, or L(p24).

* Represents a significant increase in the percentage of cells (see Section 3 for p values).

Table 2Characterization of CD4+ and CD8+ T-cells in the lymph nodes of immunized mice 3 days following the third immunizationa.

Immunogen % CM CD8 T-cells CD44hi, CD45Rbhi, CD62Lhi % EM CD8 T-cells CD44hi, CD45Rblow, CD62Llow % Eff CD4 T-cells CD44hi, CD62Llow

Naive 10.1 ± 0.2 0 6.15 ± 1.7L 9.46 0 5.56L(LA) 9.56 0 6.65p24 9.2 0 5.69L(p24) 8.97 0 4.9L(p24 + LA) 13.2 ± 1.2* 0 12.9 ± 3.9*

a Single cell suspensions were prepared from the lymph nodes of mice immunized 3 days after the second boost with either L, L(LA), p24, L(p24) or L(p24 + LA) and comparedwith age-matched control mice. The cells were stained and characterized as mentioned in Table 1. The data represents the average of two separate experiments for naiveand L(p24 + LA) immunized mice and one experiment for mice immunized with either L, (LA), p24, or L(p24), with lymph nodes pooled from three mice in all cases.

* Represents a significant increase in the percentage of cells (see Section 3 for p values).

Table 3Characterization of CD4+ and CD8+ T-cells in the PBMC of immunized mice 3 days following the third immunizationa.

Immunogen % CM CD8 T-cells CD44hi, CD45Rbhi, CD62Lhi % EM CD8 T-cells CD44hi, CD45Rblow, CD62Llow % Eff CD4 T-cells CD44hi, CD62Llow

Naive 20.35 ± 1.2 0 9.08 ± 1.2L 14.5 0 8.3L(LA) 15.5 0 10.5p24 20.5 0 14.8L(p24) 18 0 17.6L(p24 + LA) 19 ± 0.56 10.2 ± 0.38* 28.01 ± 2.1*

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.3. Cytokine profiles following in vitro stimulation with p24 or24-peptide from the spleen, lymph node, and peripheral blood of

mmunized mice

Having analyzed the effector and central memory T-cells, weext determined the cytokine profiles following in vitro stimula-ion with either p24 or the p24-peptide. Lymphocytes isolated fromhe spleens (Fig. 4A), lymph nodes (Fig. 4B), and peripheral bloodFig. 4C) of immunized mice were analyzed for a variety of cytokineshat covered both Th1/Th2 profiles. The lymphocyte cultures weretimulated with either concanavalin A as a positive control (dataot shown), p24, or p24-peptide for 48 h. The supernatants wereollected and analyzed for IL-2 and IL-4 by ELISA and for IL-6, IL-10,L-12p70, INF-�, TNF-�, and MCP-1 by the mouse CBA inflammatoryead array kit.

Splenic lymphocytes from mice immunized with L(p24 + LA) andtimulated in vitro with either p24 or the p24-peptide induced IFN-, TNF-�, IL-2, IL-6, IL-10, and MCP-1 (Fig. 4A). IL-10 and IFN-�ere the predominant cytokines produced by lymphocytes from

he lymph nodes following the second and third immunizationsFig. 4B). There was a noticeable absence of IFN-� production fromeripheral blood lymphocytes stimulated in vitro with either p24r the p24-peptide (Fig. 4C). IL-6 was present following the sec-nd immunization with L(p24 + LA), but was absent following the

(p24) or L(p24 + LA) were collected and compared with age-matched control mice.e average of two separate experiments for naive and L(p24 + LA) immunized miceooled from three mice in all cases..

third immunization (Fig. 4C). The cytokine profile shifted after thethird immunization, with MCP-1 and IL-10 being the predominantcytokines (Fig. 4C). In all three immune organs tested, IL-4 and IL-12p70 were undetectable following stimulation with either p24 orthe p24-peptide.

The cytokines induced in naive mice and in mice following thethird immunization with L, L(LA), p24, L(p24), and L(p24 + LA) afterin vitro stimulation with either p24 or with the p24-peptide areshown in Tables 4 (spleens), 5 (lymph nodes, PBMC), respectively.Splenic lymphocytes (Table 4) from mice immunized with L(p24)had a noticeable absence of IL-2 and IFN-� but secreted IL-6, IL-10, TNF-�, and MCP-1 above naive control levels. However, thecytokine production was considerably higher in mice immunizedwith L(p24 + LA). These mice also secreted high levels of IL-2 andIFN-�. Mice immunized with L, L(LA) or p24 did not induce IL-2, IL-6, IL-10, and IFN-� but produced MCP-1 at levels similar tothat observed with naive mice. Low levels of TNF-� were observedfollowing immunization with L(LA) or p24. Mice immunized withL(p24 + LA) induced p24-peptide specific T-cells to produce IL-2, IL-

6, IL-10, IFN-�, and MCP-1 in the splenic lymphocytes (lower halfof Table 4).

Lymphocytes isolated from the lymph nodes (Table 5) of miceimmunized with L(p24 + LA) secreted multiple cytokines includ-ing IL-10, IFN-�, TNF-�, and MCP-1 following in vitro stimulation

6946 N.J. Steers et al. / Vaccine 27 (2009) 6939–6949

Fig. 4. Secretion of cytokines from spleen, lymph nodes, and peripheral blood lymphocytes of mice immunized with L(p24 + LA). Single cell suspensions of (A) spleen, (B)lymph nodes, and (C) peripheral blood were stimulated in vitro with either Gag p24 or p24-peptide and the cytokines secreted were measured as described in Section 2.Both (A) spleen and (B) lymph node cells secreted IFN-�, IL-10, and MCP-1 following stimulation with either p24 or the p24-peptide. However, IFN-� was the predominantc ion. Tt werei n is af

wi

HpMo

s

ytokine secreted with the greatest induction occurring after the 2nd immunizathird immunizations. Predominantly, pro-inflammatory cytokines IL-6 and TNF-�mmunization, IL-10 and MCP-1 were the major cytokines secreted. The data showrom three mice from two separate experiments.

ith either p24 or the p24-peptide. None of the other immunogensnduced the secretion of the cytokines analyzed (Table 5).

PBMC from immune mice produced low levels of IL-6 (Table 5).owever, L(p24 + LA) was the only immunogen that induced the

roduction of multiple cytokines including IL-6, IL-10, TNF-�, andCP-1 (Table 5). Unlike spleen and lymph node cells, stimulation

f PBMCs with the p24-peptide induced low levels of MCP-1.The cytokines were undetectable or at base line levels post-

econd immunization with p24, L, or L(LA) (data not shown).

here was no difference in the levels of IL-10 or MCP-1 following the second andsecreted from (C) PBMC following the second immunization. Following the third

representative experiment of individual spleens or pooled lymph nodes and PBMC

L(p24 + LA) was the only immunogen that induced cytokineresponses post-secondary immunization (Fig. 4). The cytokine pro-files were skewed towards a Th1 type response following secondaryand tertiary immunization with L(p24 + LA) (Fig. 4).

In summary, the data demonstrated that in order to induce aneffective memory immune response, antigen alone was inadequateand that an adjuvant was required, in this case, lipid A encapsu-lated in liposomes. An increase in the population of central memoryCD8+ T-cells (CD44hi, CD45RBhi, CD62Lhi) was observed only in

N.J. Steers et al. / Vaccine 2

Table 4Cytokine production from splenic cells following stimulation with p24 or the p24-peptide following the third immunizationa.

Cytokine production (pg/mL) following p24 stimulation

IL-2 IL-6 IL-10 IFN-� TNF-� MCP-1

Naive 0 0 0 0 0 56 ± 12L(p24 + LA) 142 ± 17 88 ± 24 459 ± 51 751 ± 96 131 ± 26 693 ± 52

L(p24) 0 44 ± 8 77 ± 14 0 90 ± 9 261 ± 26p24 0 29 ± 9 0 0 55 ± 14 0L(LA) 0 0 0 0 35 ± 9 66 ± 17L 0 0 0 0 0 54 ± 9

Cytokine production (pg/mL) following p24-peptide stimulation

IL-2 IL-6 IL-10 IFN-� TNF-� MCP-1

Naive 0 0 0 0 0 0L(p24 + LA) 156 ± 26 38 ± 10 60 ± 2 340 ± 36 0 279 ± 13

L(p24) 0 36 ± 6 86 ± 16 0 0 125 ± 12p24 0 0 60 ± 11 0 0 49 ± 4L(LA) 0 0 0 0 0 57 ± 7L 0 0 0 0 0 54 ± 6

a Single cell suspensions were prepared from the spleens of mice 3 days afterthe second boost with either L, L(LA), p24, L(p24) or L(p24 + LA) and comparedwith age-matched control mice. Cells were stimulated with either p24 or the p24-peptide. Culture supernatants were collected 48 h later, clarified by centrifugation,and analyzed for cytokine secretion using either ELISA (for IL-2 and IL-12p70) ormouse inflammatory cytokine CBA assay kits, respectively. The average values ofone representative experiment out of two are shown from mice immunized withL(p24 + LA) and the corresponding naive age-matched control mice and one experi-ment from mice immunized with either L, L(LA), p24, or L(p24) with the average ofthree individual spleens in each case. For each cytokine, 300 data points were col-lected and analyzed using a FACScalibur. The mean fluorescent intensity representsthe cytokine concentration.

Table 5Cytokine production from lymph node and PBMC following stimulation with p24 orthe CTL-peptide following the third immunizationa.

Cytokine production (pg/mL) following p24 stimulation

IL-2 IL-6 IL-10 IFN-� TNF-� MCP-1

LNNaive 0 0 0 0 0 0L(p24 + LA) 0 0 624 807 92 47

PBMCNaive 0 0 0 0 0 0L(p24 + LA) 0 120 488 0 43 130

Cytokine production (pg/mL) following CTL-peptide stimulation

IL-2 IL-6 IL-10 IFN-� TNF-� MCP-1

LNNaive 0 0 0 0 0 0L(p24 + LA) 0 0 40 24 45 54

PBMCNaive 0 0 0 0 0 0L(p24 + LA) 0 0 40 0 0 40

a Single cell suspensions were prepared from the lymph nodes or the PBMC frommice 3 days after the second boost with L(p24 + LA) and compared with age-matchedcontrol mice. Lymphocytes were stimulated with either p24 or the CTL-specific-p24epitope and culture supernatants were collected 48 h later, clarified by centrifuga-tion, and measured for cytokine secretion using either ELISA (for IL-2) or mouseinflammatory cytokine CBA assay kits, respectively. No cytokines were detected inmice immunized with either L, L(LA), p24, or L(p24), except for the secretion of IL-6from lymph node cells (40 pg/mL, data not shown) in all groups. One representa-tive experiment out of two is shown from mice immunized with L(p24 + LA) andthe corresponding naive age-matched control mice. In all cases, lymph nodes andPBMC were pooled from three mice. For each cytokine, 300 data points were col-lected and analyzed using a FACScalibur. The mean fluorescent intensity representsthe cytokine concentration.

7 (2009) 6939–6949 6947

the spleen and lymph nodes while an increase in effector mem-ory CD8+ T-cells (CD44hi, CD45RBlow, CD62Llow) was seen only inthe peripheral blood following immunization with L(p24 + LA). Theinduction of effector CD4+ T-cells (CD44hi, CD62Llow) was seen inthe spleen, lymph node, and peripheral blood of L(p24 + LA) immu-nized mice. These population were absent in mice immunized withL(p24), p24, L, or L(LA). A strong Th1 type cytokine response wasinduced from lymphocytes obtained from mice immunized withL(p24 + LA) following in vitro stimulation with either p24 or thep24-peptide.

4. Discussion

We have previously shown that liposomal lipid A is apotent adjuvant for antigen processing and presentation in bothmacrophages and dendritic cells. Liposomal lipid A enhancesinflammatory cytokine production, MHC class I and class IImolecules, co-stimulatory molecules, and induces immunoprotea-somes resulting in more efficient MHC class I antigen processing[16–19]. Although, antigens encapsulated in liposomes containinglipid A have been previously reported to be presented by both MHCclass I and class II pathways [47] and to induce a strong CTL response[8,9,11,13,15] the role of liposomal lipid A in inducing effector CD4+and memory CD8+ T-cells has not been examined.

Following an acute infection, the host typically responds to thepathogen with an innate immune response, which is followed by anadaptive immune response. This leads to the generation of effectormemory T-cells, which persist within the lymphoid organs. Effectormemory T-cells are swiftly activated upon re-challenge resultingin the elimination of the pathogen [48,49]. Ideally a vaccine shouldmimic a similar adaptive immune response in terms of the induc-tion of effector memory T-cells as observed during the clearanceof an acute infection. Effector memory cells are able to migratethroughout the host and are also found in non-lymphoid tissuessuch as lung, kidney, liver, and peripheral blood and are character-ized by the low expression of CD62L [29,50,51]. Central memorycells are static and are found within the lymphoid tissue such asthe spleen and lymph nodes and are characterized by a high cellsurface expression of CD62L [52].

The functionality of CD8+ memory T-cells has been shown tobe compromised in experimental infections of CD4+ T-cell defi-cient mice resulting in the delayed clearance of the infection [53].The long-term maintenance, survival, and functionality of CD8+memory T-cells are generally dependent on CD4+ helper T-cells[54–56] and may also depend on the specific pathogen or the anti-gen [57]. Other factors such as cytokines are also essential for themaintenance and function of both effector CD4+ T-cells and mem-ory CD8+ T-cells. Furthermore, pro-inflammatory cytokines such asIFN-� and TNF-� are associated with pathogen control and clear-ance [58–61].

In the present study, we examined the role of liposomal lipidA in inducing effector CD4+ and memory CD8+ T-cell responses toHIV-1 Gag p24 in C57BL/6 mice. Our rationale for using C57BL/6mice was to further build on our recently published work [19,62].The current study indicated that central memory CD8+ T-cells wereobserved in the spleen and lymph nodes, whereas effector memoryCD8+ T-cells were found in the peripheral blood of mice immunizedwith L(p24 + LA) 3 days post-tertiary immunization. In addition, astrong induction of effector CD4+ T-cells was seen in spleen, lymphnode, and peripheral blood lymphocytes.

In general, the T-cell memory response is very rapid follow-

ing secondary exposure to an antigen or pathogen. We evaluatedthe T-cell memory response on day 3 following secondary and ter-tiary immunizations and compared it to the primary immunizationin order to capture the early induction of activated T-cells andcytokine production. Mullbacher [63] demonstrated the presence

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948 N.J. Steers et al. / Vac

f memory CD8+ T-cells with optimal lytic activity on day 3 fol-owing secondary immunization with irradiated influenza virus. Itas also been previously demonstrated that antigen-specific mem-ry CD8+ T-cells are preferentially established at 3.5 days and nott 8 days following an influenza virus challenge in mice that weredoptively transferred with antigen-specific T-cells [64].

Pro-inflammatory cytokines were seen in the spleen andymph nodes following immunization of mice with p24, L(p24)r L(p24 + LA). However, the strongest responses were observedrom mice immunized with L(p24 + LA) following in vitro stimu-ation with either p24 or the p24-peptide. The peptide stimulationndicated that the cytokine responses observed were T-cell spe-ific. Interestingly, IFN-� and TNF-� were maximally produced inhe spleen and lymph node following the second immunization.oticeably, IFN-� and TNF-� were not secreted from peripherallood mononuclear cells stimulated with either p24 or the p24-eptide. Two other cytokines that play an important role in theellular immune response, which were produced following the sec-nd immunization with L(p24 + LA), were IL-6 and MCP-1. Theseytokines were predominantly secreted by the splenic lympho-ytes and PBMC. IL-6 is an inflammatory cytokine that provideselp to transition the immune response from an innate to an adap-ive response [65]. IL-6 appears to be critical for the CD4+ memory-cell recall response as has been shown with influenza infections66]. MCP-1 attracts monocytes to the peripheral lymph nodes andpleens where they can differentiate into antigen-presenting cells67,68] and may play a role in the induction of antigen-specific

emory T-cells.IL-10 was the dominant cytokine secreted by PBMC from mice

mmunized with L(p24 + LA) following the second boost, but wasot the predominant cytokine in the spleen and lymph node.

L-10 functions as both pro-inflammatory and anti-inflammatoryytokine that can influence T-cells and professional antigen-resenting cells. The primary effect of IL-10 on T-cells is theuppression of Th1 type cytokines such as IFN-� and TNF-� [69].ndeed, we do see a decrease in the secretion of both IFN-� andNF-� from the spleen and lymph nodes of mice following ter-iary immunization with L(p24 + LA) with a concomitant increasen IL-10 secretion from PBMC. IL-10 maintains dendritic cells inn immature state [69–72] while MCP-1 recruits antigen-loadedmmature dendritic cells and monocytes to either lymph nodesr the spleen where they can undergo maturation and effectivelyresent the antigen for the priming and induction of effector mem-ry T-cells. MCP-1 was secreted from splenic lymphocytes andBMC in our studies and may have contributed to the inductionf memory T-cells.

Both CD4+ and CD8+ T-cells have the ability to produce IL-273]. IL-2 is important for the proliferation and expansion of CD8+

emory T-cells. In our study, IL-2 secretion was detected only inhe spleens of mice following the second boost with L(p24 + LA). IL-2ecretion was not detected in the lymphocytes of mice immunizedith the other immunogens. The production of IL-2 in the spleen

orrelated with the expansion of CD8+ central memory T-cells athis site. The expansion of central memory CD8+ T-cells was notbserved at sites where IL-2 was not detected.

The data presented indicates that immunization of C57BL/6ice with L(p24 + LA) induced central memory CD8+ T-cells in

he spleen and lymph nodes and effector memory CD8+ T-cellsn the peripheral blood. The induction of effector CD4+ T-cells

as observed in the spleen, lymph node, and PBMC. A predomi-antly Th-1 type of cytokine profile was observed in the spleen and

ymph nodes. IL-10 and MCP-1 were the major cytokines secretedn the PBMC. The cytokine milieu present following the immuniza-ion with L(p24 + LA) appears to be supportive for the induction offfector CD4+ and memory CD8+ T-cells. Furthermore, our studiesemonstrate the requirement of the adjuvant liposomal lipid A for

[

7 (2009) 6939–6949

the induction of effector and memory T-cells, which secrete Th-1 type cytokines upon exposure to antigen. Thus, the inclusion ofliposomal lipid A as an adjuvant with a candidate vaccine may beessential to induce an effective memory T-cell response and suchstudies will be carried out in the future utilizing higher animalspecies.

Acknowledgements

This work was supported by an in-house-laboratory indepen-dent research award from the Walter Reed Army Institute ofResearch. We would like to thank Ms. Elaine Morrison for help withthe animal studies.

Conflict of interest: The authors have no financial conflicts ofinterest.

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