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Immunization with Th-CTL Fusion Peptide and Cytosine-Phosphate-Guanine DNA in Transgenic HLA-A2 Mice Induces Recognition of HIV-Infected T Cells and Clears Vaccinia Virus Challenge ¹

Pirouz Daftarian^*, Saima Ali^*, Rahul Sharan^*, Simon F. Lacey^*, Corinna La Rosa^*, Jeff Longmate, Christopher Buck, Robert F. Siliciano and Don J. Diamond^2,*

^* Laboratory of Vaccine Research and Division of Information Sciences, Beckman Research Institute of the City of Hope, Duarte, CA 91010; and Division of Medicine, Johns Hopkins University Medical School, Baltimore, MD 21218

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We evaluated immunogenicity of a novel Th-CTL fusion peptide composed of the pan DR Th epitope and a CTL epitope derived from HIV-pol in two transgenic HLA-A*0201/K^b mouse models. The immunogenicity of peptides of this structure is highly dependent on coadministered cytosine-phosphate-guanine DNA. Initial evaluations of peptide-specific immunity are based on results of chromium release assay, intracellular cytokine, and tetramer staining. Significant cytotoxic T cell responses are found upon a single immunization with as low as 0.1 nmol both peptide and cytosine-phosphate-guanine DNA. Splenocytes from immunized mice recognize naturally processed HIV-pol expressed from vaccinia virus (pol-VV). Translation of immunologic criteria into more relevant assays was pursued using systemic challenge of immunized mice with pol-VV. Only mice receiving both peptide and DNA together successfully cleared upward of 6 logs of virus from ovaries, compared with controls. Challenge with pol-VV by intranasal route of intranasal immunized mice showed a significant reduction in the levels of VV in lung compared with naive mice. A convincing demonstration of the relevance of these vaccines is the robust lysis of HIV-infected Jurkat T cells (JA2/R7/Hyg) by immune splenocytes from peptide- and DNA-immunized mice. This surprisingly effective immunization merits consideration for clinical evaluation, because it succeeded in causing immune recognition and lysis of cells infected with its target virus and reduction in titer of highly pathogenic VV.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Potent CTL responses are critical in the immunological defense against HIV infection (1). Several studies have correlated elevated HIV-specific CTL responses with resistance to infection (2, 3, 4), long-term nonprogression to AIDS (5), and protection against lentivirus challenge in animal models (6). Recently, investigators have begun using the growing number of defined HLA-restricted minimal CTL epitopes from several HIV gene products as a novel strategy to induce potent antiviral CTL responses (7). One potential approach is the administration of a DNA plasmid encoding several minimal epitopes linked together as a string of beads under the control of a constitutive promotor (8). Alternatively, synthetic peptides have been made that correspond to as many as 10 CTL epitopes, and their activity in mouse models has been excellent (9).

CTL epitopes can be delivered as part of synthetic chimeric peptide constructs (10, 11, 12, 13). Powerful immunological adjuvants or chemical modification have traditionally been used to augment the CTL response to such peptides (12, 13). Unfortunately, many adjuvants either fail to induce Ag-specific CTLs, or have associated side effects that make them unsuitable for human use (13, 14, 15). Recently, bacterial and synthetic DNA have attracted much attention as potentially safe and effective adjuvants for human use, with the capacity to promote potent CTL responses following parenteral (16) or mucosal administration (17, 18). Their mechanism of action in stimulating T cell adaptive immunity appears indirect, and is initiated by interaction with pathogen-associated molecular pattern receptors such as Toll-like receptor 9 on the surface of APCs (19). Their ability to up-regulate Th1 responses contributes to augmenting the function of a variety of vaccines, including those for hepatitis B, herpes simplex II, HIV, and peptides (20, 21). Cytosine-phosphate-guanine (CpG) ³-containing DNA exert immunoregulatory effects at a number of distinct steps in the immune response cascade, and the sum total of the immunomodulatory effects is to enhance the activity of vaccines targeting cellular immunity.

This study is an investigation of the use of a sequence-conserved, HLA-A*0201-restricted synthetic CTL epitope constructed into a fusion peptide as a model HIV vaccine approach (22). The pol_464–472 was selected, because of the extensive published background on its structure, expression, and usage (23, 24). It was shown to be a high affinity binder to HLA-A*0201, and a significant fraction of chronically infected HIV/AIDS patients continue to recognize it, even when they are undergoing highly active antiretroviral therapy (7, 25, 26). When formulated with IFA, we and others have found that minimal sequence HLA-restricted CTL epitopes function poorly as immunogens, reflecting the lack of Th required to drive and maintain the CTL response (27, 28). Exogenous Th can be provided in trans by using the pan HLA-DR-binding epitope (PADRE) (29); however, the vaccine still requires formulation with a potent adjuvant to evoke a cytolytic response (28). In this work, transgenic (Tg) HLA-A2/K^b mice were used to investigate the immunogenicity of the minimal HLA-A*0201-restricted HIV clade B CTL epitope pol_464–472 in combination with PADRE as a single fusion peptide solely composed of both epitopes. When administered in saline and formulated with CpG DNA, the fusion peptide conferred potent stimulation of splenic CTL, whether administered s.c. or intranasally (i.n.). Murine CTL recognized endogenously processed reverse transcriptase (RT) expressed from vaccinia virus (VV), lysed T cells infected with HIV, while immunized mice conferred protection to challenge by pol-VV. These results suggest that epitope fusion peptides could potentially be used as a vaccine in highly active antiretroviral therapy-treated HIV/AIDS patients, delivered either by mucosal or parenteral routes.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Peptides

The HLA-A*0201-resticted CTL epitopes from HIV pol_464–472 (I9V), human CMV tegument protein pp65 (pp65_495–503, N9V), the hepatitis B nucleocapsid Ag (core_18–27, F10V), the human p53 gene (p53_149–157, S9V), and the promiscuous Th epitope PADRE were synthesized individually on the Applied Biosystems 432 (Foster City, CA) or as PADRE-CTL epitope fusion peptides using standard Fmoc procedures (see Table I) by manual synthesis. Single epitope peptides were determined to be 90% pure; fusion peptides were 85% pure by HPLC analysis; and correct molecular masses (±2 atomic mass units) were confirmed by matrix-associated laser desorption/ionization-time of flight using a Kompact Probe (Kratos Analytical, Chestnut Ridge, NY).

Synthetic oligodeoxynucleotide (ODN) 1826 with CpG motifs underlined (5'-TCCATGACGTTCCTGACGTT-3') and non-CpG ODN 1982 (5'-TCCAGGACTTCTCTCAGGTT-3') (30) were synthesized with nuclease-resistant phosphorothioate backbones by DNA (Montreal, Québec, Canada). The Na⁺ salts of the ODNs were resuspended at 5 mg ml^-1 in 10 mM Tris (pH 7.0), 1 mM EDTA, and stored as 50-µl aliquots at -20°C before dilution in aqueous 0.9% sodium chloride solution before injection.

Animals and immunization schedule

All immunizations were conducted using 8- to 12-wk-old Tg HLA-A*0201/K^b (hereafter A2/K^b) or HHDII mice bred onto the C57BL/6 background obtained from a breeding colony maintained at the City of Hope (Duarte, CA) (28, 31, 32). Both mouse models contain a transgenic HLA-A*0201 molecule, whose native 3 domain is replaced with the corresponding 3 domain of the murine H-2K^b (28) or D^b (32) molecule, which provides increased levels of T cell responsiveness in these Tg models. For parenteral administration, animals received peptide vaccine as a single s.c. injection at the base of the tail in a final volume of 100 µl saline. For i.n. administration, peptide solution was applied as droplets over the external nares of anesthetized animals (30 ml/kg of ketamine/xylazine cocktail (Sigma-Aldrich, St. Louis, MO)) in a final volume of 30 µl saline. Rectal immunization was conducted by depositing the vaccine solution into the colon of anesthetized (as described above) animals via the anus using a Gilson P200 pipette and sterile yellow tip in a final volume of 30 µl. Three mice were immunized per group, unless otherwise stated.

In vitro expansion of CTLs

Spleens were retrieved from sacrificed animals, and a single cell splenocyte suspension was prepared by passing the cells through a 70-µm Falcon cell strainer (BD Labware, Franklin Lakes, NJ) using the plunger from a sterile 1-ml syringe. Splenocytes were subjected to one round of in vitro stimulation (IVS) for both peptide and virus recognition, as recently described (33). Splenocytes from immunized animals were cocultured with peptide-loaded LPS blasts in complete IVS medium at a ratio of 3:1 for 7 days, with the addition of 10% rat T-stim (Collaborative Biomedical Products, Bedford, MA).

Cytotoxicity assay

Cytolytic activity was determined using a 4-h chromium release assay (CRA) after 1 IVS with the following modifications (34). T2 cells (TAP-deficient human cell line) (35) and Jurkat A2.1 with or without cotransfected HIV plasmid were used as targets (36). Cells were cultured in human target medium, as recently described (34). Jurkat A2.1 transfectants were maintained with 0.25 mg/ml of Geneticin or 0.25 mg/ml hygromycin B (Life Technologies-BRL, Rockville, MD) in human target medium. T2 target cells (American Type Culture Collection, Manassas, VA) in log phase were pulsed with 10 µM of the relevant or nonrelated synthetic peptide for 1 h. Jurkat A2.1 cells were infected for 2–3 h at multiplicity of infection of 15 with VV constructs, according to published protocols (37). Both T2 and Jurkat A2.1 cells were labeled with 200 µCi of Na ⁵¹CrO₄^- (ICN, Costa Mesa, CA) for 1 h in a 37°C water bath, and further processed, as described (34). Experimental evaluations were performed in triplicate, and assay data were acceptable if background was <30% for peptide-loaded targets, and the average and SD were <15% of the mean.

CRA statistics

Cytotoxicity was regarded as significant relative to controls at a given E:T ratio if all three experimental mice had higher percentage of cytotoxicity than all control mice, corresponding to p = 0.05 by a one-sided Wilcoxon test. Cytotoxicity was regarded as significant overall (denoted by an asterisk in figures) if it was significant at the highest two E:T ratios, corresponding to p = 0.0025 (unless otherwise indicated in the figure legend).

Measurement of p24 Ag in JA2/R7/Hyg cells

Culture supernatants of HIV-1-infected JA2 cells (JA2/R7/Hyg) and uninfected JA2 cells were collected, filtered through a 0.2-µM acetate filter, and analyzed for p24 by ELISA. HIV replication was assessed in cell-free supernatant by ELISA (HIV-1 p24 ELISA kit; NEN Life Science, Boston, MA), as per manufacturer’s instructions. Levels of p24 were measured in cell-free supernatants sampled every 2–3 days.

HLA-A*0201 (A2) tetramers

HLA-A2 tetramers were produced in our laboratory using a minor modification of the procedure used by the National Institute of Allergy and Infectious Diseases Tetramer Core Facility (http://www.emory.edu/WHSC/TETRAMER/protocol.html). HLA-A*0201 H chain and ₂-microglobulin (₂m) were produced from Escherichia coli XA90 following transformation with the pHN1 constructs. Recombinant H chain and ₂m were refolded in the presence of either the I9V or N9V peptide (33, 38).

mAbs and flow cytometric analysis

Intracellular cytokine (ICC). Intracellular IFN- expression was detected using Cytofix/Cytoperm Plus Kit with GolgiStop (BD PharMingen, San Jose, CA). Splenocytes either ex vivo or after 1 wk IVS (2 x 10⁶/ml) were plated in 96-well, round-bottom plates and stimulated with 5 µg/ml I9V or S9V as irrelevant peptide for 6 h. Staining was assessed by FACSCalibur (BD Biosciences, San Jose, CA), and data were analyzed using CellQuest software for MacIntosh.

Tetramer. Anti-murine CD8 FITC was purchased from BD PharMingen. Staining and washing were performed using FACS buffer (PBS containing 0.5% FCS and 0.1% sodium azide). For direct fluorescent labeling, 1 million cells were incubated with either A2-I9V or A2-N9V tetramer (1 µg per sample in 20 µl) for 30 min at 4°C, and washed with cold FACS buffer. Cells were then incubated with anti-CD8 FITC (2 µl per sample in 20 µl) at 4°C for an additional 30 min. After completing the staining process, cells were again washed, then analyzed immediately using a FACSCalibur flow cytometer. Data were analyzed using CellQuest software for MacIntosh.

Construction of a ubiquitinated pol gene for detecting CTL activity against full-length RT

The human ubiquitin (Ub) gene followed by the Arg codon and e^k sequence (39, 40) was amplified using the following pairs of primers (5' primer A, CTTAAgCTTggTgCggCCgCCATgCAgATCTTC, and 3' primer B, TAATACTgACgCTCgAgCgggCCCTCgggAAAC). The PCR conditions were 94°C, 2 min; 25 cycles of 94°C, 30 s, 65°C, 30 s, 72°C, 40 s, and followed by 72°C for 1 min. The resulting 363-bp Ub-R-e^k PCR product was gel purified and cloned into the pSC11-MCS (pSC11) insertion plasmid (modified with a polylinker) using NotI and ApaI restriction enzyme sites to generate Ub-R-e^k-pSC11 (41, 42, 43). The HIV-1 pol gene containing both reverse-transcriptase (RT) and integrase (In) protein domains was extracted and amplified from pNL4-3 (plasmid containing full-length clone of clade B HIV-1) (44) using the following two primers (5' primer C, TTgATCgggCCCATTAgCCCTATTgAgACTgTACCA, and 3' primer D, gAAggCCTCTAATCCTCATCCTgTCTACTTgCCAC). The PCR conditions were 94°C, 2 min; 25 cycles of 94°C, 30 s, 62.2°C, 30 s, 72°C, 4 min, and followed by 72°C for 10 min. The 2544-bp PCR product was gel purified and cloned into Ub-R-e^k-pSC11 using ApaI and StuI restriction sites to produce Ub-R-e^k-RT-In-pSC11 (Ub-R-pol). The nonubiquitinated form was made into a VV by directly cloning the 2544-bp PCR product into pSC11 (pol-VV). Plasmid constructs were verified by restriction enzyme digestion and DNA sequencing. Ub-R-pol-VV was generated by transfecting the Ub-R-pol-pSC11 plasmid into wild-type VV-infected Hu thymidine kinase^- cells, as described (37). Ub-R-pol-VV was subjected to four rounds of simultaneous screening and selection using color reaction of substrates (Bluogal; Sigma-Aldrich) to -galactosidase and resistance to 5-bromo-2'-deoxyuridine. The expression of Ub-modified RT-In and unmodified RT-In expressed from vCF21 was detected by Western blot using mAb21 (data not shown) made available through the National Institutes of Health AIDS Research and Reference Reagent Program (45). The unmodified HIV-1 clade B pol-expressing VV (vCF21) was expanded from seed stock obtained from the National Institutes of Health AIDS Research and Reference Reagent Program (46). All VV were expanded in suspension HeLa cell culture, as described by Moss and Earl (47). Virus of sufficient titer (10¹⁰ PFU) was applied to sucrose gradients and purified of cellular debris, first by pelleting, then banding, and retitered before use for in vivo challenge studies.

In vivo VV challenge assay

Mice (female) were immunized with two doses of the vaccine formulation (100 µl/mouse for each immunization) on days 0 and 14 by s.c. and i.p. administration, respectively. Seven days after the second immunization, the mice were challenged i.p. with 1 x 10⁷ PFU of VV-expressing Ub-R-pol, or Ub-M-gag, or nonubiquitinated pol-VV. Five days after the challenge, the mice were sacrificed and the ovaries were removed for VV titer measurements, as described (48). Mucosal challenge was conducted after two doses of vaccine were administered in the nares separated by the same period as for parenteral immunizations. A total of 2 x 10⁷ PFU of Ub-R-pol-VV was administered to the nares in 30 µl PBS, after light anesthesia (30 ml/kg of ketamine/xylazine cocktail (Sigma-Aldrich)). Five days later, the mice were sacrificed, and both lungs were removed. Following homogenization and three rounds of freeze-thaw, the ovaries or lungs were assayed for VV. The titer of VV in the ovaries or lungs was determined by plating serial 10-fold dilutions of the homogenized ovaries or lungs on a six-well plate of CV-1 cells. Following overnight incubation, the cells were stained with crystal violet and the plaques were counted at each dilution. The minimal detectable level of the virus was 100 PFU/ml.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Th-CTL fusion peptide vaccine: immunogenicity without additional adjuvant

To investigate whether the immunogenicity of the Th and CTL epitopes could be increased by their administration as a single molecule, a strategy that has been used successfully with other CTL and/or Th epitopes (9, 49, 50, 51), we synthesized a fusion peptide consisting of the I9V CTL epitope covalently attached to the COOH terminus of the PADRE Th epitope (Table I). No additional amino acid residues were included in the PADRE:I9V fusion peptide sequence to separate the two linked epitopes, as the PADRE molecule contains three alanine residues at the carboxyl terminus that could serve as a natural spacing motif (52, 53). The CTL response was investigated in HLA-A2/K^b mice inoculated with 1–100 nmol PADRE:I9V fusion peptide in saline (data not shown). It required 100 nmol fusion peptide to obtain 48% specific lysis of I9V peptide-pulsed targets after a single round of IVS at an E:T ratio of 100 (data not shown). This response was dose dependent, as activity was diminished to background levels when the dose of fusion peptide was reduced to 50 nmol or less (data not shown). To obtain more robust immunogenicity from the fusion peptide, a strategy using CpG DNA was evaluated.

CpG DNA augments the immunogenicity of Th-CTL fusion peptide

To assess whether the inclusion of CpG DNA would increase the cytolytic responses to the fusion peptide vaccine, HLA-A2/K^b mice were immunized once via the s.c. route with molar equivalents of ODN 1826 (CpG DNA) or 1982 (non-CpG DNA) and PADRE:I9V fusion peptide as a mixture in saline. Fourteen days later, spleens were retrieved and CTL responses were determined after one round of IVS. As can be seen in Fig. 1, administration of 1–20 nmol of both PADRE:I9V fusion peptide and ODN 1826 resulted in substantial levels of peptide-specific cytotoxicity at all E:T ratios tested. These responses ranged from 8- to 13-fold higher than the cytolytic responses observed at the same E:T ratios when only 0.05 nmol fusion peptide + CpG DNA was administered (Fig. 1). Compared with the group in which mice were administered 3.8 nmol (25 µg) of CpG 1826 alone, all vaccine doses of 0.1 nmol or higher showed statistically significant increases in peptide-specific cytotoxicity (Fig. 1). No cytolytic activity above background was noted in any group, when target cells were loaded with irrelevant N9V peptide, or mice were immunized with fusion peptide alone, or with non-CpG DNA (data not shown).

View larger version (15K): [in this window] [in a new window]   FIGURE 1. PADRE:I9V fusion peptide requires CpG DNA to be immunogenic at low concentration. Tg HLA-A2/Kb mice were immunized s.c with equal amounts of PADRE:I9V and CpG DNA. After 14 days, splenocytes from individual mice were stimulated in vitro for 7 days with irradiated I9V-loaded LPS blasts. Cytolytic T cell responses were determined by CRA using T2 cells loaded with I9V peptide as targets. Levels of cytotoxicity against irrelevant N9V-loaded T2 cells at the same E:T ratios for all vaccine doses were always at or below CpG DNA control levels using I9V peptide-loaded targets. *, p < 0.05 vs CpG DNA alone.

To more precisely quantify Ag-specific CD8⁺ T cells within the lymphocyte population, we used HLA tetramers containing either the I9V or irrelevant N9V (33, 54) (data not shown) minimal CTL epitopes to track peptide-specific CD8⁺ T cells (see Materials and Methods). In vitro cultures derived from a separate group of Tg mice immunized with 25 nmol PADRE:I9V fusion with 3.8 nmol CpG DNA, in a similar manner as described in Fig. 1, contained a subset of CD8⁺ T cells (18.35% of total CD8⁺ cells), which were capable of binding the HLA-A2-I9V tetramer (Fig. 2aiii). In contrast, <1.0% of CD8⁺ cells obtained from a similar culture originating from animals administered CpG DNA without peptide could bind the HLA-A2-I9V tetramer (Fig. 2aii). A comparable background level of 0.3% was observed when both cultures were stained with an HLA-A2 tetramer containing the unrelated N9V epitope (Table I), although only the CpG DNA + peptide culture is shown (Fig. 2a, compare i and iii). The ability of these CD8⁺ cells to bind HLA-A2-I9V tetramer was also associated with potent lytic activity. Effector CTL obtained from mice immunized with both fusion peptide and CpG DNA were very efficient killers, with 80% lysis observed at an E:T ratio of 100 (data not shown).

View larger version (42K): [in this window] [in a new window]   FIGURE 2. Tetramer (HLA-A2-I9V) binding after IVS. a, Mice were immunized with 3.8 nmol CpG DNA (ii) or 25 nmol PADRE:I9V and 3.8 nmol CpG (i, iii). After 14 days, splenocytes from individual mice were stimulated in vitro for 7 days with irradiated I9V-loaded LPS blasts. A total of 1 x 106 splenocytes was stained with CD8 FITC and I9V-tetramer-PE. Cells were washed and analyzed using the FACSCalibur. Data analysis was performed using CellQuest, and the results are presented as dot plots. i, Represents the N9V-tetramer-PE control. b, ICC staining after IVS. Mice were immunized with equal amounts of PADRE:I9V and CpG, either 1 nmol (iii, iv) or 10 nmol (ii, v, vi, vii). After 14 days, splenocytes from naive mice (i) and immunized mice were stimulated in vitro for 7 days with irradiated I9V-loaded LPS blasts. These cells were then incubated for 6 h with either I9V or irrelevant peptide. The cells were passed through Ficoll-Hypaque gradient, and the live cells were washed with PBS. A total of 1 x 106 cells was stained with CD8 FITC. The cells were then fixed and permeabilized by Perm/Fix (BD PharMingen) solution for 30 min at 4°C. Cells were washed in permeabilizing buffer and incubated with either anti-IFN- PE (i, iii–vii) or isotype-matched PE-labeled mAb (ii). Samples were analyzed using the FACSCalibur. Data analysis was performed using CellQuest, and the results are presented as dot plots.

Intracellular IFN- results after IVS

We examined whether this vaccination approach stimulates both lytic function as well as cytokines, which are both presumably important in successful vaccine strategies against HIV infection. To establish whether cytotoxic function (see Fig. 1) is related to the frequency of IFN--secreting T cells, ICC assays were conducted after IVS and restimulation with I9V or S9V peptide (Table I) for 6 h, after which cells were stained to detect intracellular IFN-. Immunized mice (s.c.) that were examined for cytotoxicity in Fig. 1 were also examined by ICC assay (Fig. 2b). Compared with naive mice, which had sporadic positive cells (Fig. 2bii), two mice immunized with 1 nmol peptide and DNA showed between 6.8 and 19% IFN--secreting CD8 T cells (Fig. 2b, iii and iv). In the case of the 10 nmol immunization, there was greater than a doubling of the I9V peptide-specific IFN--secreting T cells to an average of 30% (Fig. 2b, v–vii). There was negligible reactivity to an isotype control Ab (Fig. 2bi), or the nonspecific peptide S9V (data not shown). Similarly, when mice were immunized with CpG DNA without peptide, there was little recognition of I9V (data not shown). The results of ICC staining agree with those of CRAs in Fig. 1.

Fusion peptide vaccine formulation is immunogenic via the mucosal route

The major portals of entry for numerous infectious agents are the mucosal surfaces of the genitourinary, gastrointestinal, and respiratory tracts. An effective vaccine against infection by such pathogens, including HIV, should have the potential to induce both systemic and mucosal immune responses. To evaluate the ability of the fusion peptide vaccine to induce a systemic CTL response following mucosal delivery, 50 nmol PADRE:I9V with 3.8 nmol ODN 1826 was administered via the i.n. route. This experimental series was repeated twice, using three mice in each instance. In the absence of CpG DNA, the administration of a single i.n. dose of fusion peptide failed to induce peptide-specific cytolysis following one IVS of splenocytes with syngeneic I9V-loaded LPS blasts (data not shown). In contrast, the addition of 3.8 nmol CpG DNA to the vaccine formulation markedly enhanced the resultant CTL activity in splenocyte cultures (Fig. 3). In the presence of CpG DNA, cytotoxicity was increased 50-fold compared with the response against fusion peptide alone at an E:T ratio of 100 after a single administration (data not shown). The booster immunization enhanced cytolytic function (Fig. 3). Note that regardless of the number of immunizations, the nonspecific recognition of the S9V peptide is very limited. Immunogenicity of the fusion peptide vaccine is similarly up-regulated in both the systemic and i.n. routes of immunization. These results corroborate work conducted with other fusion peptides that were sensitive to the inclusion of CpG DNA for cytolytic response via the mucosal route (33).

View larger version (15K): [in this window] [in a new window]   FIGURE 3. CTL responses following mucosal administration of fusion peptide. Tg HLA-A2/Kb mice were immunized i.n. on day 0 with 50 nmol PADRE:I9V peptide and 3.8 nmol CpG DNA. One group of mice (second) was immunized again i.n. on day 14 with 50 nmol PADRE:I9V peptide and 3.8 nmol CpG DNA. Fourteen days following the second immunization, pooled splenocytes from three mice were stimulated in vitro for 7 days with irradiated I9V-loaded LPS blasts. Cytolytic T cell responses were determined by 51Cr release assay using T2 cells loaded with I9V peptide or S9V peptide as targets.

Immunologic studies with HIV Ags as full-length proteins have more relevance to in vivo infection vs in vitro recognition of processed CTL epitopes. We chose to use a more efficient system for recognition of HIV proteins, using the ubiquitinated forms of the Ag. Previously, we have shown that ubiquitinated forms of CMV-pp65 are better recognized than the native form by CTL effectors (33). Therefore, we evaluated the RT-In gene that is enhanced for recognition as a result of fusion of the Ub cassette to the N terminus earlier described by Varshavsky (55). The results shown in Fig. 4 are after two s.c. immunizations with 50 nmol HIV vaccine fusion peptide along with 3.8 nmol CpG DNA. The data shown are representative of three additional experiments. The control cell line, JA2, elicits minimal recognition, which is significantly less than the cell line infected with Ub-R-pol-VV at all E:T tested (Fig. 4). In contrast, recognition of pol-VV-infected targets is minimal (data not shown), most likely because of the less efficient processing leading to epitope generation, when the Ag is not modified with Ub (33, 39). Similar data from mucosal immunizations agree with the results shown for s.c. immunization (data not shown).

View larger version (12K): [in this window] [in a new window]   FIGURE 4. Recognition of naturally processed HIV-pol expressed from VV. Tg HLA-A2/Kb mice were immunized s.c. on days 0 and 14 with 50 nmol PADRE:I9V and 3.8 nmol CpG DNA. On day 21, splenocytes from individual mice were stimulated in vitro for 7 days with irradiated I9V-loaded LPS blasts. Cytolytic T cell responses were determined by CRA using either JA2 cells (squares) or Ub-R-pol-VV-infected JA2 cells as targets. *, Difference between infected and uninfected cells is significant at p < 0.003.

It is important to establish whether the fusion peptide vaccine is capable of protecting against a viral infection, despite favorable immunogenicity parameters. Immunocompetent rodent models of HIV infection have traditionally involved the use of VV or other viruses that infect mice. In experiments shown in Fig. 5a, mice were immunized s.c. and i.p. with 50 nmol HIV fusion peptide vaccine and 3.8 nmol DNA (i, ii), or DNA alone (v). In one set of experiments, only a single immunization was conducted using a total of 10 nmol peptide and CpG DNA (iii), but the results are substantially the same as with mice given two doses of fusion peptide (i, ii). The use of combined s.c. and i.p. injection to evaluate protection is based upon work published by Berzofsky and collaborators (56). Challenge experiments were replicated three times, and each panel in Fig. 5 illustrates results of one representative experiment.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Fusion peptide immunization results in protection from VV challenge in Tg mouse model. a, Tg HLA-A2/Kb mice were immunized s.c. on day 0 and i.p. on day 14 either with 3.8 nmol CpG DNA (v), or with 3.8 nmol CpG DNA and 50 nmol PADRE:I9V peptide (i, ii). On day 21, the mice were challenged i.p. with rVV. A total of 1 x 107 PFU of rVV expressing either pol (i, iv) or Ub-R-pol (ii, v). After 5 days, the virus titer in the ovaries was determined. For group iii, the mice were immunized with 5 nmol PADRE:I9V peptide and 5 nmol CpG DNA via both the s.c. and i.p. routes on day 0. On day 14, the mice were challenged i.p. with 1 x 107 PFU of pol-VV. After 5 days, the virus titer in the ovaries was deter mined. *, Significant at p < 0.03. b, Tg HLA-A2/Kb mice were immunized s.c. on day 0 and i.p. on day 14 with 3.8 nmol CpG DNA (iii, iv), or with 3.8 nmol CpG DNA and 50 nmol PADRE:I9V peptide (i, ii). On day 21, mice were challenged with 1 x 107 PFU rVV expressing either Ub-R-pol or Ub-M-gag. After 5 days, the virus titer in the ovaries was determined. *, Significant at p < 0.03. c, Groups of three mice were immunized i.n., either with 10 (iii) or 1.0 (iv) nmol PADRE:I9V and CpG DNA or 3.8 or 10.0 nmol CpG DNA alone (ii-a, b). On day 14, all groups (i–iv) were challenged i.n. with 2 x 107 PFU pol-VV. After 5 days, the virus titer in the lungs was determined. d, Mice were immunized s.c. on day 0 and i.p. on day 14 with either 10 nmol both PADRE:I9V and CpG DNA (), or 10 nmol CpG DNA alone (). On day 21, the mice were challenged with pol-VV, as indicated on the x-axis. After 5 days, the virus titer in the ovaries was determined. Value of p < 0.01 between CpG-immunized mice vs PADRE:I9V/CpG-immunized mice summed for all challenge doses.

VV expressing alternative Ag is not protected by HIV fusion peptide

We addressed whether a challenge infection by a VV encoding a different Ag than RT-In would be cleared by fusion peptide immunization, to rule out nonspecific effects exerted by presentation of vaccinia-expressed Ags. In a similar manner to the experiment in Fig. 5a, mice were immunized with the fusion peptide and CpG DNA or DNA alone. Mice were given two identical doses of the vaccine in a 2-wk period, and 7 days after the second dose, they were challenged with 10 million PFU of different viruses, including those that expressed HIV RT-In or those that expressed HIV-gag (Fig. 5b). All viruses expressed Ags in enhanced presentation formats using the Ub system, as described in Materials and Methods. In a consistent and statistically significant manner, the experiment shows that only a combination of the HIV fusion peptide vaccine and subsequent challenge by Ub-R-pol-VV resulted in successful protection from challenge by 6 logs (i). Mice immunized with DNA alone (iii, iv) or challenged with a heterologous virus (ii, iv) were not protected from challenge.

Mucosal immunization with fusion peptide protects against mucosal VV challenge

We also used the nares as a challenge route to simulate a possible route of HIV infection via a mucosal surface (18). Groups of three mice were immunized into the nares on days 0 and 14 with 3.8/10 nmol CpG DNA, or 1 and 10 nmol CpG DNA and PADRE:I9V peptide in a minimal volume. Similar to the parenteral immunization, 1 wk after the second immunization, 20 million PFU of rVV was introduced in the nares. After 5 days, the virus titer in the lungs was determined. Evident from Fig. 5c is a 5- to 6-log difference in protection between mice that were immunized with CpG DNA alone, vs those that were immunized with 10 nmol CpG DNA and fusion peptide vaccine. An additional experiment, in which the vaccination took place intrarectally and the challenge dose of VV was administered i.n., showed an approximate 4-log reduction of VV titer in the lungs (data not shown). Accompanying the challenge study was a flow cytometry analysis of HLA tetramer-positive CD8 T cells after the second booster immunization. In the CpG DNA-immunized animals, only 0.1% of the CD8 T cells bound the A2-I9V tetramer, whereas the addition of 10 nmol fusion peptide increased the level to 1.6% of the CD8 T cells binding to the tetramer (data not shown). Similarly, in mice that received an intrarectal (i.r.) immunization followed by i.n. challenge, an average of 1.3% of the CD8 T cells bound the I9Vtetramer (data not shown). This experiment demonstrates the possibility of control of a VV mucosal infection by mucosal administration of the fusion peptide vaccine.

Titration of VV challenge: limits to protection against viral challenge

What level of challenge virus would be required to overcome the effects of systemic immunization using the HIV fusion peptide vaccine? A titration series was conducted between 1 and 50 million PFU of challenge virus administered i.p. into mice that had been previously immunized, either with fusion peptide and DNA vs DNA alone. The experiment shown in Fig. 5d shows that a challenge virus dose lower than 10 million PFU is insufficient to reliably infect the Tg mouse strain used in our study. Between 10 and 20 million PFU, and similar to the experiments shown in Fig. 5, a and b, there is almost a 6-order magnitude difference between animals that were immunized with the combination of peptide and DNA vs those immunized with DNA alone. Finally, at the 50 million PFU challenge, two of three mice were protected in the peptide plus DNA-immunized group vs no mice protected when only CpG DNA was administered. These data indicate that a substantial amount of protection is provided by the peptide vaccine, even at the 50 million PFU challenge dose .

Novel HIV in vitro recognition model: p24 expression in JA2/R7/Hyg cells

Because HIV is unable to infect the rodent model directly, an alternative strategy is to examine the capacity of immune cells from the immunized mouse to recognize HIV-infected human T cells. A well-described model exists in which an HIV provirus has been transfected into the Jurkat human T-thymoma model, as described by Tsomides et al. (59). Human HIV-specific CTL recognize this T cell target of HIV infection (data not shown) (59). Jurkat T cells were transfected with an HLA-A2.1 expression plasmid, and an infectious plasmid DNA clone expressing nef-deleted HIV (pHXB2). We first demonstrated that these T cells make the p24 Ag, an accepted indicator of HIV infection. The Jurkat T cells, referred to as JA2/R7/Hyg (R7), were put into culture, and after 2 days, medium samples were taken for ELISA. Shown in Fig. 6a are the results of measurements of p24 Ag from R7 cells after dilution with fresh complete medium. The included p24 standard at 0.25 ng/ml had a similar OD reading as the 1/5 dilution of the R7 medium. Note that measurements of medium from JA2 cells had levels of p24 indistinguishable from the substrate control. The difference in p24 Ag levels was statistically significant between JA2 and R7 cell lines (p < 0.05). This measurement confirms that the HIV provirus in R7 cells is functional, and CTL lysis of R7 would be indicative of recognition of HIV-infected human T cells by murine splenocytes.

View larger version (14K): [in this window] [in a new window]   FIGURE 6. In vitro recognition of HIV-infected JA2 (R7) cells by splenocytes from peptide + DNA-immunized Tg HLA-A2/Kb mice. a, Culture supernatants of R7 (1:5, 10) and JA2 (negative) cells were collected and filtered through a 0.2-µM acetate filter. HIV replication was assessed in cell-free supernatant by ELISA (HIV-1 p24 ELISA kit; NEN Life Science), as per manufacturer’s instructions. Levels of p24 were measured in cell-free supernatants sampled every 2–3 days. Bars identified as Std and Substrate were assays done with material supplied by the manufacturer. b, Mice were immunized s.c. with either 1 or 0.1 nmol both PADRE:I9V and CpG DNA. After 14 days, splenocytes from individual mice were stimulated in vitro for 7 days with irradiated I9V-loaded LPS blasts. Cytolytic T cell responses were determined by CRA using either JA2 or R7 cells as targets. *, Significant at p < 0.05.

We have adapted this model to our mouse immunization system by conducting in vitro recognition experiments. Mice immunized twice by the s.c. route with HIV fusion peptide vaccine and DNA were sacrificed 1 wk after the final immunization, and splenocytes were subjected to one round of IVS, and then tested against the R7 or parental JA2 cell line (Fig. 6b). After two immunizations with low dose of peptide vaccine and DNA, there is a striking difference between recognition of the parent JA2 cell line vs the HIV provirus-expressing R7 line (Fig. 6b). Immunization with as low as 0.1 nmol peptide vaccine and DNA results in a statistically significant difference in cytolytic recognition vs the parent JA2 cell line (Fig. 6b). The recognition of the R7 cell line after 1 nmol immunization is striking, and it demonstrates the vigorous immune response to the peptide vaccine that is generated in the HLA-A2/K^b Tg mouse model.

HHDII mice have enhanced recognition of naturally processed HIV RT

Recently, Lemonnier and colleagues (32) developed an alternative Tg model that expresses the human HLA-A2.1 molecule, but is deficient in mouse ₂m and the H-2D^b gene. The animals are deficient in mouse MHC class I and II cell surface expression, but express human ₂m because it is covalently attached to the HLA-A2/K^b trangene. It more accurately reflects expression of HLA structures in the context of a humanized background without competing expression of endogenous mouse MHC. These animals were immunized with 50 nmol HIV fusion protein either with 3.8 nmol DNA (group 1) or alone without DNA (group 2). Animals were immunized twice, and after 1 cycle of IVS, they were evaluated for recognition of HIV Ag, including I9V nominal peptide (Fig. 7a), followed by VV-expressing full-length HIV RT (Fig. 7b) and finally R7 cells (Fig. 7c). CTL responses to the vaccine in the HHDII mice have similarities and differences with the recognition evaluated in the Tg HLA-A2/K^b model. The response to peptide-sensitized T2 cells is very similar (compare Fig. 1), but the cytolytic response to Vac-infected JA2 cells (Fig. 4) and R7 cells (Fig. 6b) is marginally better in the HHDII mice. In each of the Ag evaluations, there is a statistically significant difference between animals that are immunized with peptide plus DNA (group 1) vs animals immunized with peptide alone. Especially noteworthy are the recognition of full-length RT expressed in VV (Fig. 7b) and the HIV-infected R7 cell line (Fig. 7c). These robust responses are validation that the peptide vaccine is inducing significant immune responses in a Tg animal that more closely simulate the human immune response than other models (60).

View larger version (13K): [in this window] [in a new window]   FIGURE 7. HHD mice have enhanced recognition of naturally processed HIV-pol Ag. HHD mice were immunized s.c. on days 0 and 14 either with 50 nmol PADRE:I9V and 3.8 nmol CpG DNA (group 1) or with 50 nmol PADRE:I9V alone (group 2). On day 21, splenocytes from individual mice were stimulated in vitro for 7 days with irradiated I9V-loaded LPS blasts. Cytolytic T cell responses were determined by CRA. a, CRA was performed using T2 cells loaded with I9V peptide as targets. *, Significant at p < 0.003. b, CRA was performed using JY cells infected with Ub-R-pol-VV. *, Significant at p < 0.05. c, CRA was performed using HIV-expressing R7 cells. *, Significant at p < 0.003.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The simplicity of delivering synthetic peptides to vertebrates has made them attractive vaccine candidates, and methods to enhance their immunogenicity have been continuously developed and tested in the last 15 years. Although minimal CTL epitopes have been shown to stimulate correlates of immunity, comparable success using in vivo immunization to protect against or treat viral infection has been less promising (75, 76, 77, 78). The early enthusiasm for delivery of minimal CTL epitopes as immunologic signals that represent full-length HIV proteins has been tempered with the realization that the effectiveness of immune responses is dependent upon enhancement of both the CD4⁺ and the CD8⁺ arms of the cellular immune system (50, 79, 80). Two general rules have emerged that are crucial for success. They include providing a mechanism that enhances Th, and an adjuvant that is best able to enhance a Th1 milieu, such as the recently described CpG phosphorothioate ODN (81, 82).

To increase immunogenicity and vaccine efficacy of peptidic CTL epitopes, investigators have considered multiple immunization strategies. These include alteration of peptide structure and inclusion of adjuvants and cytokines in vaccine formulations, as mechanisms to enhance CD4⁺ Th and durability of immune responses (13, 83). Structural modifications have included various forms of lipidation as means to enhance the recognition of the peptide CTL epitope (37, 84). A noninclusive list of formulations includes cofactors that have been introduced along with the peptide, such as inflammatory cytokines (56, 85, 86), cholera and E. coli labile toxin (13, 87), tripalmitoyl-s-glycerylcysteine (88, 89), or traditional adjuvants (IFA, QS-21, Montanide, MF-59, etc.) to provide a favorable milieu for immunologic recognition. Various delivery strategies to deliver minimal CTL epitopes, based on using either long chimeric peptides, naked DNA, or viruses, have been evaluated (9, 90, 91, 92). The relative complexity of these delivery and formulation strategies in contrast to approaches that rely on enhancement of innate immunity and Th1 bias makes the newer approaches using CpG DNA more attractive.

We have previously evaluated chimeric fusion peptides that are significantly hydrophobic, using a noncognate Th epitope called PADRE, in combination with coadministered CpG DNA (33). We systematically investigated the two criteria of providing CD4⁺ Th and adjuvant on vaccine efficacy in this study using the model peptide PADRE:I9V (Table I). It was confirmed that the covalent attachment of the CTL to the Th epitope conveyed an increased level of recognition (data not shown) that was further enhanced by the inclusion of CpG DNA (Figs. 1 and 7). Immunization with CpG DNA alone did not stimulate recognition of the I9V CTL epitope (Fig. 1). In fact, there was a remarkable uniformity of response to the peptide and DNA formulation between 1 and 20 nmol (Fig. 1). In experiments that we have not shown, a dose titration of CpG DNA starting at 0.01 nmol enhanced recognition of PADRE:I9V, and greater amounts do not change sensitivity (data not shown). We also obtained comparable results with a fusion peptide composed of PADRE and the S9L peptide that was formulated in the identical manner with CpG DNA (data not shown).

We investigated two different strategies of formulation of peptide and CpG DNA; either equimolar of each reactant, or variable amounts of peptide and constant amounts of CpG DNA. When the amount of CpG DNA in the formulation exceeded 1 nmol, it positively impacted both immunogenicity and protection assays. This strategy compares well to experiments in which PADRE:I9V was emulsified in IFA. Experiments repeated several times showed 50% of the activity with IFA compared with CpG DNA in combination with the peptide in saline (data not shown). Because IFA provided little or no advantage to the much less inflammatory adjuvant, CpG DNA, we elected not to further pursue experiments involving IFA emulsions. This decision is based on the lack of approval of IFA for human use, and the favorable characteristics of CpG DNA for eventual clinical use (82, 93).

Studies from several laboratories in the last 12 years have examined peptides as immunogens. The majority of the studies have come from the Berzofsky Laboratory in which both immunologic correlates and protection against VV challenge in mice have been evaluated (48, 56, 79, 83, 85). Our formulation provides for higher levels of vaccine efficacy than previous reports, including correlates of immunity and protection from viral challenge, using CpG DNA as adjuvant. Certainly, not all vaccine systems and strategies are directly comparable with our report. We have presented consistent data demonstrating that correlates of immunity using functional cytotoxicity assays (Fig. 1) are in agreement with quantitative flow cytometry measures using both tetramers (Fig. 2a) and IFN- release (Fig. 2b). Peptide-specific responses were translated into a more meaningful measurement of recognition, when targets were infected with HIV-pol-VV that expresses the RT Ag (Fig. 4). Ubiquitination of the Ag was meant to develop a system in which the success of a vaccine could be more readily measured using an Ag presentation format that is more effective than the expression of the full-length protein without modification (33). This experiment established that the peptide vaccine stimulated lymphocytes that were able to recognize naturally processed Ag, a key criteria for evaluation of an HIV vaccine. In preliminary experiments, we established that pol-VV and gag-VV were less efficient than the ubiquitinated forms to direct recognition of JA2 APCs (data not shown). This method of vaccine analysis has proven useful in both CMV and HIV studies, and it can be used in any infectious disease or cancer immunotherapy system in which the immune response to Ags requires quantitation. A recent report demonstrated that Ub-enhanced proteasomal processing made a DNA-based CTL epitope cancer vaccine more efficacious (94).

Immune correlates establish whether a vaccine can generate immunity, but protection against viral infection or suppression of viremia are generally considered more physiologic parameters of the efficacy of the vaccine. We pursued that aspect of vaccine efficacy, by evaluating whether immunization with HIV fusion peptide would be able to suppress an ongoing VV infection. Preliminary experiments established that the peptide vaccine administered without CpG DNA was incapable of diminishing viremia, which is in line with its relative incapability of stimulating an active T cell response (data not shown). In contrast, and consistent with the immune correlates that were measured, the fusion peptide vaccine in combination with the CpG DNA was effective at eliminating an infection initiated with a 20 million PFU dosage of HIV-pol-VV. This protection occurred whether the pol-VV was modified with Ub, or was presented as a native Ag (Fig. 5a). This result was unexpected, based on the in vitro parameters of recognition, requiring the ubiquitinated form of pol-VV (Fig. 4). It is possible that protection from virus challenge is measuring a different immunologic function than CTL recognition. Originally, 50 nmol fusion peptide vaccine in combination with 3.8 nmol CpG DNA were administered as an s.c. prime, followed by an i.p. boost 2 wk later. Remarkably, a dose of 10 nmol peptide + DNA given once and divided equally between s.c. and i.p. routes was as effective as the larger dose given twice (Fig. 5a). Because it is known that CpG DNA are capable of attenuating some bacterial and viral infections (58, 95, 96), we also showed that the same amount of CpG DNA without coadministered peptide given in the prime/boost immunizations did not attenuate the VV infection (Fig. 5a).

Nonspecific attenuation of a VV expressing a different HIV Ag than RT-In was also explored. The result showed that the specificity of the peptide vaccine was only for the RT-In Ag (Fig. 5b). Similar to earlier work reported by Ahlers et al. (79), we showed that the challenge dose could be increased to 50 million PFU of VV, and we still obtained a significant level of protection in two of three mice tested (Fig. 5d). At all doses, protection was dependent upon coadministration of both CpG DNA and the HIV fusion peptide vaccine. The level of protection is statistically significant. More importantly, the minimal dosing of the vaccine combined with the significant levels of protection (>6 logs) suggest that the delivery strategy and vaccine formulation are an important new addition to the repertoire of molecules that should be further evaluated for efficacy in primates or humans.

A more physiologic model than protection against VV infection would address protection against HIV infection, although such a model does not yet exist in immunocompetent rodents. To address the important issue, we used a novel in vitro model. We first demonstrated that HIV-infected R7 Jurkat cells made p24 Ag by traditional ELISA used for quantitating HIV infection (Fig. 6a). In the original description of the cell line that we confirmed, a human HIV-specific T cell clone that recognized the I9V peptide also efficiently recognized and killed R7 cells (59) (data not shown). For the first time, we have shown that splenocytes from HLA-A2/K^b mice immunized with as little as 0.1 nmol HIV peptide vaccine and CpG DNA recognized R7 T cells, and the 1.0 nmol dose caused substantial recognition (Figs. 6b and 7c). This study establishes this cell model as an additional in vitro method to evaluate HIV vaccines.

HIV entry frequently occurs via the mucosal route, especially in cases of sexually transmitted infection (97). Therefore, we investigated both systemic and mucosal routes of immunization to establish whether both were significant portals for administration of the HIV fusion peptide vaccine. Previously, it was shown that either i.n. or i.r. administration of a Th:CTL fusion peptide will elicit both systemic and mucosal T lymphocytes that can protect against infection by VV in rodent models (48). A similar protocol using peptides that matched SIV immunogenic sequences conveyed protection against simian HIV in a macaque model (13). We demonstrated that the same vaccine formulation that was effective systemically in eliciting CTL and providing protection against infection in the ovary was effective using the i.n. route of administration for protection against infection of the lungs. The effectiveness of the peptide vaccine for protection was found to be more limited than the equivalent vaccine applied s.c. (Fig. 5, compare a and c). Mucosal protection is also dependent on simultaneous administration of both peptide and CpG DNA. In a pilot study, we also demonstrated that administration of the peptide vaccine with DNA via an i.r. portal was as effective as the i.n. route at eliciting CTL, and similar levels of protection were found with both routes (data now shown).

These studies support further evaluation of these types of peptide vaccines, especially in the context of CpG DNA, which confers upon them robust activity in eliciting CD8⁺ CTL and protection against VV infection. As earlier indicated, the partial independence from Th responses for efficacy by vaccines formulated with CpG DNA makes them attractive for use in immunosuppressed individuals, such as HIV/AIDS patients (81). In summary, peptide vaccines based upon combination of the Th epitope PADRE and CTL epitopes from either the pol or gag genes are an effective means to elicit HIV-specific immunity in Tg rodent models. This strategy has important elements of efficacy that have been documented as a result of the killing of T cells that are infected with HIV. As with many other vaccine candidates, more stringent tests in primates and humans will be necessary to fully appreciate whether this type of vaccine would be an effective candidate for evaluation to prevent HIV infection.

	Acknowledgments

 
The technical assistance of John Brewer, Shahram Mekhoubad, and Peiling Wang is gratefully acknowledged. We acknowledge the early work of Dr. John Simms establishing the immunogenicity of the fusion peptide. We thank the staff of the Animal Resources Center at City of Hope for excellent care of research animals and maintenance of breeding colonies. We acknowledge F. Lemonnier of the Pasteur Institute for providing us with a breeding colony of HHDII mice, and J. Primus (Vanderbilt University, Nashville, TN) for use of the HLA-A2/K^b Tg mice. We acknowledge the National Institutes of Health AIDS Research and Reference Reagent Program for providing the JA2/R7/Hyg cells, mAb 21, and vCF21. We acknowledge Dr. Bernard Moss (Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases) for the pSC11 plasmid, Dr. Bruce Walker (Massachusetts General Hospital, Charlestown, MA) for the human T cell clone 68A62, and Dr. John Rossi (City of Hope) for the pNL4-3 plasmid. We thank Beckman Coulter Immunomics (San Diego, CA) for provision of plasmids enabling us to construct HLA-A*0201 tetramers. We thank Drs. Belyakov and Berzofsky for helpful instructions in carrying out mucosal and systemic virus challenge experiments. The assistance of Donna Packer and Julia Santos in the administration of the laboratory and preparation of the manuscript has greatly aided the completion of this work.

	Footnotes

 
¹ These studies have been partially supported by grants from the National Institutes of Health, Division of AIDS (RO1-AI43267 and R21-AI44313), the National Cancer Institute (RO1-CA77544 and PO1-CA30206, Project III), and University-wide AIDS Research Program (BRI-054) to D.J.D. The City of Hope Comprehensive Cancer Center is supported by the National Cancer Institute (CA33572). D.J.D. is partially supported by a Translational Research Award from the Leukemia and Lymphoma Society, and the Laboratory of Vaccine Research is partially supported by The Edwin and Bea Wolfe Charitable Foundation.

² Address correspondence and reprint requests to Dr. Don J. Diamond, Laboratory of Vaccine Research, Beckman Research Institute of the City of Hope, City of Hope Medical Center, 1500 E. Duarte Road, Duarte, CA 91010. E-mail address: ddiamond{at}coh.org

³ Abbreviations used in this paper: CpG, cytosine-phosphate-guanine; ₂m, ₂-microglobulin; CRA, chromium release assay; ICC, intracellular cytokine; i.n., intranasal; In, integrase; i.r., intrarectal; IVS, in vitro stimulation; ODN, oligodeoxynucleotide; PADRE, pan DR epitope; RT, reverse transcriptase; Tg, transgenic; Ub, ubiquitin; VV, vaccinia virus.

Received for publication April 8, 2003. Accepted for publication August 14, 2003.

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