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Increased Level and Longevity of Protective Immune Responses Induced by DNA Vaccine Expressing the HIV-1 Env Glycoprotein when Combined with IL-21 and IL-15 Gene Delivery¹

Elizabeth Bolesta^*, Aleksandra Kowalczyk^*, Andrzej Wierzbicki^2,*, Cheryl Eppolito^*, Yutaro Kaneko, Masafumi Takiguchi, Leonidas Stamatatos, Protul A. Shrikant^* and Danuta Kozbor^3,*

^* Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263; Institute of Immunotherapy for Cancer, Kinki University, Osaka, Japan; Division of Viral Immunology, Center for AIDS Research, Kumamoto University, Kumamoto, Japan; and Seattle Biomedical Research Institute, Seattle, WA 98109

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
We investigated the ability of a plasmid-derived IL-21 delivered alone or in combination with the IL-15 gene to regulate immune responses to the HIV-1 envelope (Env) glycoprotein induced by DNA vaccination. Mice were injected with the gp140CFI_HXB2/89.6 vector expressing a modified Env glycoprotein with C-terminal mutations intended to mimic a fusion intermediate, in which the most divergent region encoding the variable V1, V2, and V3 domains of CXCR4-tropic HxB2 virus was replaced with the dual-tropic 89.6 viral strain. Using a recombinant vaccinia virus expressing 89.6 Env glycoprotein (vBD3) in a mouse challenge model, we observed that IL-21 plasmid produced sustained resistance to viral transmission when injected 5 days after DNA vaccination. Moreover, IL-21 in a synergistic manner with IL-15 expression vector augmented the vaccine-induced recall responses to the vBD3 challenge compared with those elicited by immunization in the presence of either cytokine alone. The synergistic combination of IL-21 and IL-15 plasmids promoted expansion of CD8⁺CD127⁺ memory T cell pools specific for a subdominant HLA-A2-restricted Env_121–129 epitope (KLTPLCVTL). Our results also show that coimmunization with IL-21 and IL-15 plasmid combination resulted in enhanced CD8⁺ T cell function that was partially independent of CD4⁺ T cell help in mediating protection against vBD3 challenge. Furthermore, the use of IL-21 and IL-15 genes was able to increase Ab-dependent cellular cytotoxicity and complement-dependent lysis of Env-expressing target cells through augmentation of Env-specific IgG Ab levels. These data indicate that the plasmid-delivered IL-21 and IL-15 can increase the magnitude of the response to DNA vaccines.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The current challenge for the design of an effective HIV-1 vaccine is to develop immunization strategies to elicit both broader and sustain immune responses against diverse viral species. Such vaccines include those in which protective immune responses are stimulated by live, recombinant viruses, or DNA-based vectors (1, 2, 3, 4, 5, 6, 7), and augmented by a variety of cytokines or costimulatory molecules (8, 9, 10, 11, 12, 13, 14, 15, 16). Among cytokines that are of particular interest for the generation of an efficacious HIV-1 vaccine are those that share the common receptor chain (_c)⁴ subunit and are implicated in the process of memory cell generation (17, 18, 19). Among them, IL-2, IL-4, IL-7, IL-15, and IL-21 are all involved in T cell responses and some of these cytokines are required for the development or maintenance of memory T cells (18, 20, 21, 22, 23, 24). IL-2, one of the most extensively studied cytokine that is produced by CD4⁺ T cells, drives rapid clonal expansion of naive T cells leading to their subsequent differentiated state (18), but can also limit clonal expansion and the accumulation of Ag-specific effector cells by promoting activation-induced cell death (25). IL-7 is crucial for the survival and homeostatic expansion of naive CD8⁺ T cells and can also contribute to memory CD8⁺ T cell homeostasis (22, 26, 27, 28). IL-15, another survival factor, exerts its effect primarily on memory CD8⁺ T cells (29, 30) by stimulating their proliferation rather than apoptosis and promoting memory T cell turnover (21, 24, 31, 32, 33). IL-15 can also provide a costimulus to induce B cell proliferation and Ig secretion (34). In the context of immune therapy and genetic vaccine, IL-15 has been shown to be effective against tumors as well as some infectious disease models (13, 35, 36, 37, 38, 39).

IL-21, the last member of the IL-2 cytokine family that exhibits functional overlaps with IL-2 and IL-15, has been suggested as an effective adjuvant in enhancing and sustaining CD8⁺ T cell responses (40, 41). It efficiently promotes proliferation, cytotoxic activity, and IFN- production by murine and human CD8⁺ effector T cells (41, 42). IL-21 also induces activated NK cell terminal differentiation and functions, thereby limiting NK cell expansion by IL-15 (43). In addition, a severe defect in IgG1 production following Ag priming in IL-21R-deficient mice (44), indicates a role for IL-21 in regulating Ab production (44, 45, 46). In view of these findings, it has been suggested that IL-21 can influence both innate and adaptive immunity (43, 45). In fact, IL-21 has recently been shown to increase survival rates of HSV-1-infected mice when coinjected with a plasmid DNA encoding HSV-1 glycoprotein B Ag (47), and mice treated with IL-21 have been able to reject tumor cells and develop tumor-specific immune responses (40, 48, 49, 50).

Considering the dominant role that CD4⁺ T cells and IL-2 cytokine family play in the generation and maintenance of HIV-1-specific immune responses, it seems important to better characterize the role of IL-21 in regulating the size of naive and memory T cell pools in response to HIV-1 Ags in vivo. This information is likely to facilitate the development of a rational approach for improving the efficacy of potential HIV-1 vaccines. In this study, we have investigated the ability of a plasmid-encoded IL-21 delivered alone or in combination with the IL-15 gene to regulate the survival of envelope (Env)-specific CD8⁺ T cell memory pool as well as the level and isotypes of Ab responses induced by the gp140CFI_HXB2/89.6 DNA expression vector in mice. We report here that IL-21 plasmid in a synergistic context with IL-15 gene augmented expansion of the vaccine-induced memory CD8⁺ T cells and mediated more vigorous recall responses to the vBD3 vaccinia virus challenge than animals immunized in the presence of either cytokine alone. The synergistic combination of IL-21 and IL-15 also promoted expansion of CD8⁺CD127⁺ memory T cell pools specific for the subdominant HLA-A2-restricted Env_121–129 epitope (KLTPLCVTL) (2), and has resulted in enhanced CD8⁺ T cell function that was partially independent of CD4⁺ T cell help in mediating protection against vBD3 challenge. The interaction between these cytokines also increased Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against Env-expressing target cells predominantly through augmentation of Env-specific IgG Ab levels.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Construction of the HIV-1 chimeric HxB2/89.6 Env DNA vaccine

The codon-optimized gp140CFI_HxB2 Env glycoprotein cloned in the VRC2801 expression vector (provided by Dr. G. Nabel, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD) was used for generation of the HIV-1 chimeric gp140CFI_HxB2/89.6 construct (4). Briefly, gp140CFI_HxB2 was first subcloned to the pBluescript II SK⁺ vector using BamHI and XbaI restriction enzymes sites. Subsequently, two sites NsiI and Bsu36I were introduced at positions 105 and 363, respectively, using a QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s procedure. After DNA sequence verification, the plasmid was digested with NsiI and Bsu36I restriction enzymes and the entire fragment from residues 105 to 363 in gp140CFI_HxB2 was replaced with the corresponding sequence from 89.6 Env (accession number U39362) to create the Env chimeric construct. Then, the entire fragment between BamHI and XbaI restriction sites was cloned into the VRC2801 expression vector. After the second DNA sequence verification, the expression of the gp140CFI_HxB2/89.6 gene was confirmed by immunoprecipitation of the Env glycoprotein from culture supernatants of radiolabeled 293T cell transfectants.

Transfection, labeling, and immunoprecipitation

Transient transfection of 293T cells with plasmid DNA encoding either the gp140CFI_HxB2 or gp140CFI_HxB2/89.6 gene was performed using Lipofectamine Reagent (Invitrogen Life Technologies) according to the manufacturer’s protocol. For the radioimmunoprecipitation analysis, [³⁵S]methionine/cysteine (400 µCi/plate; DuPont-New England Nuclear) was added on the second day of transfection for an additional 24-h period. At the end of the incubation period, culture supernatants were collected, concentrated 100-fold on a Millipore filter (Millipore), and immunoprecipitated with a mixture of sera from HIV-1-infected individuals (51) followed by polyclonal rabbit anti-human Ig (ICN Biomedicals) and protein A-Sepharose CL-4B (Pharmacia Biotech). The immunoprecipitates were separated by Blue Native PAGE (BN-PAGE) as described (52). Typically, 5 µg of purified proteins was loaded per lane, and the gels were fluorographed to visualize [³⁵S]methionine/cysteine-labeled proteins.

Immunization of rabbits

Female New Zealand rabbits were immunized with the Env vaccine at the Lampire Biologicals. Briefly, each rabbit received two i.m. gp140CFI_HxB2/89.6 DNA injections (500 µg per rabbit) in a 3-wk interval followed by three protein boosts. For the booster immunization, soluble gp140CFI_HxB2/89.6 Env glycoprotein was purified from culture supernatants of 293T transfectants by lectin affinity chromatography, emulsified with IFA, and injected (500 µg per rabbit) s.c. on days 42, 63, and 84. Before immunization, the level and integrity of the purified Env were confirmed by the BN-PAGE analysis. The immune sera were collected on day 94 and checked for reactivity with the Env glycoprotein by an ELISA. Preimmunized sera were included as negative controls.

Env-pseudotyped virus neutralization and luciferase reporter cell assay

The assay was performed as previously described (53, 54). Briefly, a HeLa cell line that expresses CD4, CXCR4, and CCR5 was used as target cells for HIV-1 infection. These cells, initially called JC53-bl and now termed TZM-bl, were obtained from the National Institutes of Health AIDS Reference and Reagent Repository (Germantown, MD). The cells contain Tat-responsive reporter genes for firefly luciferase and Escherichia coli -galactosidase under regulatory control of an HIV-1 long-terminal repeat. In the assay, the level of HIV-1 infection was quantified by measuring relative light units (RLUs) of luminescence that are directly proportional to the amount of virus input.

One day before the neutralization assay setup, 3000 TZM-bl cells were plated in a 96-well flat-bottom tissue culture plate in 100 µl of complete medium. Dilutions of heat-inactivated (56°C for 1 h) sera from pre- and postimmunized rabbits were made in duplicate in 96-well round-bottom tissue culture plates. A predetermined amount of pseudovirus that resulted in 2 to 5 x 10⁵ RLUs was added to the diluted sera for 1.5 h at 37°C. In separate wells, cells were incubated with virus in the absence of the Abs. During the incubation, the TZM-bl cells were treated with polybrene (2 µg/ml) for 30 min at 37°C. The medium containing polybrene was aspirated from the cells and replaced with 50 µl of fresh medium combined with 50 µl of the virus/serum mixture and incubated for 3 days at 37°C. Cell lysis occurred by replacing the medium with 100 µl of 1x Cell Culture Lysis Reagent (Promega). Cells were lysed for 1 h at room temperature and subjected to one freeze-thaw cycle. Then 60 µl of the lysate was loaded onto a corresponding well in a Black Enhanced Binding 96-well solid plate, a luciferase substrate (Promega) was added to each well (100 µl /well), and the RLUs associated with each well were recorded with a Fluoroskan Ascent FL (Thermo Labsystems). Percent neutralizations were calculated as previously described (54).

Recombinant vaccinia viruses (rVVs)

The rVVs expressing the full-length Env of HIV-1_89.6 (vBD3) (55) and -galactosidase (vSC8) (56) were provided by Drs. R. Collman (University of Pennsylvania, Philadelphia, PA) and B. Moss (Laboratories of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD), respectively.

Mice and immunization

Six-week-old BALB/c (H-2^d) mice were purchased from The Jackson Laboratory. The HLA-A2/K^b-transgenic mice, expressing hybrid molecules bearing the 1 and 2 domains of HLA-A2.1 fused to the 3 domain of H-2K^b, were provided by Dr. L. Sherman (The Scripps Research Institute, San Diego, CA). All mice were maintained in a specific pathogen-free microisolator environment, and the experimental procedures were performed in compliance with protocols approved by the Institutional Animal Care and Use Committee of the Roswell Park Cancer Institute. Mice were injected i.m. in both quadriceps with the gp140CFI_HxB2/89.6 vector (10, 30, 100, or 300 µg/mouse). The immunization was repeated three times in a 3-wk interval. Control mice were immunized with the sham plasmid. The IL-21- or IL-15-encoded pORF9 vector (10, 20, or 40 µg/mouse; InvivoGen) was delivered i.m. at the time of vaccination with the gp140CFI_HxB2/89.6 construct. Two additional groups of gp140CFI_HxB2/89.6-immunized mice received IL-21 plasmid on day 5 or 15 after vaccination. The optimal dose of the IL-21 and IL-15 expression vectors that were used in combination with the gp140CFI_HxB2/89.6 vaccine was determined using the vBD3 vaccinia virus challenge study.

Protection against challenge with the vBD3 vaccinia virus

At different time points after the last DNA immunization, mice were challenged i.p. with 2 x 10⁷ PFU of vBD3 or vSC8 rVV. Five days later, mice were sacrificed, ovaries removed, homogenized, sonicated, and assayed for vaccinia virus titer by plating serial 10-fold dilutions on human HuTK^– 143B indicator cells, staining with crystal violet and counting plaques at each dilution as described (2). To evaluate the contribution of CD8⁺ and CD4⁺ T cells as well as NK cells to the vaccine-induced protection against the vBD3 challenge, gp140CFI_HxB2/89.6-immunized mice were treated i.p. with 100 µg of anti-CD4 mAb (clone GK 1.5), anti-CD8 mAb (clone 53-6.72), or with 25 µl of anti-asialo GM1 (GA1) rabbit serum (Wako Chemicals) on days 3 and 1 before and day 1 after the challenge as described (57, 58). To investigate whether IL-15 and IL-21 would enhance the gp140CFI_HxB2/89.6 vaccine-induced protection against vBD3 challenge in mice that were partially depleted of CD4⁺ or CD8⁺ T cells, BALB/c mice were injected i.p. with anti-CD4 or anti-CD8 mAb before and during the immunization period. The Ab treatment was capable of depleting CD4⁺, CD8⁺, or NK cells in nonimmunized mice by 90% as determined by a flow cytometric analysis with anti-CD4 (clone H129.19), anti-CD8 (clone 53-5.8), and CD49b/Pan-NK cells (clone DX5) mAbs (BD Pharmingen). In parallel, control groups of gp140CFI_HxB2/89.6-immunized mice were treated with 100 µg of rat IgG (ICN Biomedicals).

Cytotoxicity assay

Splenocytes from BALB/c mice were cultured at 2 x 10⁶ cells/ml in 24-well culture plates with vBD3-infected monocyte-macrophage cell line PU5–1.8 (American Type Culture Collection) and 15% T cell stimulatory factor (T-STIM Culture Supplement; Collaborative Biomedical Products) as a source of exogenous IL-2. Before incubation, vBD3-infected stimulator cells were incubated with 0.5% paraformaldehyde on ice to inactivate the virus (51), washed, and mixed with splenocytes at a 1:40 ratio. After 3 days of stimulation, cells were split and cultured in medium supplemented with 0.3 ng/ml recombinant mouse IL-2 (BD Pharmingen). The cytolytic activity was analyzed 6 days later by a standard 4-h ⁵¹Cr release assay against P815 cells infected with vBD3. Control target cells were infected with vSC8. The percent specific lysis was calculated as: ((cpm experimental release – cpm spontaneous release)/(cpm maximum release – cpm spontaneous release)) x 100. Maximum release was determined from supernatants of cells that were lysed by addition of 5% Triton X-100. Spontaneous release was determined from target cells incubated with medium only.

ELISPOT assay

The numbers of Env-specific and IFN--secreting CD8⁺ splenocytes in gp140CFI_HxB2/89.6-immunized and control mice were determined by the ELISPOT assay as described (59). Briefly, 96-well nitrocellulose plates (Millipore Multiscreen-MAIP; Millipore) were coated with 15 µg/ml rat anti-mouse mAb directed to IFN- (MAB785; R&D Systems) in 0.05 M carbonate-bicarbonate buffer (pH 9.6). After overnight incubation at 4°C, the wells were washed with PBS containing 0.05% Tween 20 and blocked for 1 h with RPMI 1640 medium containing 10% FCS. For the analysis of frequencies of Env-specific and IFN--secreting CD8⁺ T cells, splenocytes were combined with vBD3-infected P815 cells at a ratio of 3:1, and placed in 2-fold dilutions into the Ab-coated wells. For each dilution, duplicate samples were used. After 24 h of incubation at 37°C, the plates were washed six times with PBS containing 0.05% Tween 20 and incubated for 2 h with 50 µl of 1 µg/ml biotinylated mAb directed to mouse IFN- (MAB485; R&D Systems). The plates were washed and incubated for 1 h with 50 µl of 1/1000 diluted streptavidin-conjugated alkaline phosphatase (SA-5100; Vector Laboratories). After final wash with PBS, spots were developed with an alkaline phosphatase 5-bromo-4-chloro-3-indolyl-1-phosphate (BCIP)/NBT substrate (SK-5400; Vector Laboratories) and counted under a stereomicroscope. The frequencies of IFN--secreting cells were determined by regression analysis from a curve generated by plotting the number of spots vs the number of effector cells.

HLA-tetramer analysis

MHC class I tetramers folded with the subdominant HLA-A*0201-restricted Env_121–129 epitope (KLTPLCVTL) (2) was prepared as previously described (60). Splenocytes harvested from gp140CFI_HxB2/89.6-immunized HLA-2/K^b-transgenic mice were incubated with Fc Block (anti-CD16/32) for 10 min and then labeled with PE-Env_121–129/HLA-A*0201 tetramer and anti-CD8-CyChrome mAb in combination with either anti-CD44-FITC, anti-CD62L-FITC, or anti-CD127 (IL-7R)-FITC mAb (BD Pharmingen). The cells were washed twice in HBSS containing 0.01% NaN₃ and 1% FCS, fixed with 1% paraformaldehyde, and stored at 4°C in the dark before acquisition. Background staining was assessed using an isotype control Ab. The number of Env_121–129/HLA-A*0201 tetramer-specific CD8⁺ T cells secreting IFN- was determined using BD Cytofix/Cytosperm kit and allophycocyanin-conjugated IFN- mAb (BD Pharmingen) according to the manufacturer’s protocol. All flow cytometric evaluations were performed on FACScan or FACSCalibur flow cytometer. After gating on forward and side scatter parameters, at least 10,000-gated events were routinely acquired and analyzed using CellQuest software (BD Biosciences).

Measurement of Env-specific Ab end point titers

To detect serum Abs against the Env glycoprotein by Con A ELISA, plates were coated with 100 µl of Galanthus nivalis lectin (Sigma-Aldrich) (10 µg/ml) overnight at 4°C. The lectin solution was removed from the wells and blocked with 200 µl of PBS containing 10% FBS for 2 h at room temperature. The plates were washed twice with PBS containing 0.2% Tween 20 (PBST), and then coated with the Env glycoprotein purified on a lectin-column from supernatant of vBD3-infected 293T cells. The plates were washed with PBST five times, and then sera from different groups of the immunized and cytokine-treated mice were added in 3-fold dilutions. After a 1-h incubation at room temperature, the plates were washed five times and incubated with a 1/1000 dilution of alkaline phosphatase-conjugated goat anti-mouse IgM or IgG (Sigma-Aldrich) in PBST. Subisotyping of serum IgG Ab responses in the immunized mice was conducted with alkaline phosphatase-conjugated rat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 secondary Abs (BD Pharmingen). After washing, the reaction was developed with p-nitrophenylphosphate (1 mg/ml; Bio-Rad) in diethanolamine buffer (Bio-Rad), stopped with 0.4 M NaOH, and analyzed at 405 nm with an ELISA plate reader Dynatech MRX. Sample dilutions were considered positive if the OD recorded for that dilution was at least 2-fold higher than the OD recorded for the control sample at the same dilution (61).

ADCC and CDC assays

For the ADCC assay, different dilutions of sera from the immunized mice were incubated for 6 h with ⁵¹Cr-labeled P815 cells infected with vBD3 or vSC8 vaccinia virus together with NK cell-enriched splenocytes isolated from naive BALB/c mice. NK cells were enriched by centrifugation over a discontinuous density gradient consisting of 70, 65, 60, 57, 55, and 50% Percoll (Amersham Biosciences) as described (62), and recovered in the lower density fractions (63).

The CDC against cells expressing the Env glycoprotein was assayed at serum dilutions of 1/30, 1/90, and 1/270 with rabbit complement (Cedarlane Laboratories) by a standard ⁵¹Cr release assay against vBD3-infected P815 cells as described (64). Cells incubated only with medium, complement, or antisera served as controls. Spontaneous release was calculated based on the chromium released by target cells incubated with complement alone. Maximum radioactivity release was determined from supernatants of cells that were lysed by the addition of 5% Triton X-100. The percent of specific lysis was calculated as: ((cpm experimental release – cpm spontaneous release)/(cpm maximum release – cpm spontaneous release)) x 100.

Statistical analyses

The significance of differences in the number of Env-specific CD8⁺ T cells and Env-specific Ab titers in different groups of the immunized mice was determined using a two-tailed Student’s t test assuming equal variance. Mixed-model ANOVA (65) was used to compare mean values of the vBD3 viral titers between control mice and those immunized with gp140CFI_HxB2/89.6 in the presence or absence of IL-15 and IL-21 plasmids. The p values for the pairwise group comparisons for the average vBD3 virus titers in the immunized mice that were challenged over the 6-mo period were computed using the nonparametric Wilcoxon’s rank-sum test. Data were presented as arithmetic mean ± SD and analyzed using the JMP software (SAS Institute) on a Windows-based platform.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Expression of the gp140CFI_HxB2/89.6 mutant and induction of neutralizing Ab responses

For the DNA vaccination study, we have expressed the CCR5-tropic version of the gp140CFI_HxB2/89.6 Env glycoprotein on the backbone the codon-optimized gp140CFI_HXB2 construct, which has been previously modified to reduce gp160-induced cytotoxicity, stabilize the formation of trimers, and eliminate formation of the hairpin intermediate (4). In the gp140CFI_HxB2/89.6 mutant, the most divergent region encoding aa 105–363 in gp140CFI_HXB2 was replaced with the corresponding sequence from the 89.6 Env glycoprotein (Fig. 1A). After DNA sequence verification, the oligomeric state of the secreted gp140CFI_HxB2/89.6 Env glycoprotein in culture supernatants of transiently transfected 293T cells was analyzed by immunoprecipitation and BN-PAGE. Supernatants from cells transfected with the gp140CFI_HXB2 construct (Fig. 1B, lane 2) and the sham plasmid (Fig. 1B, lane 1) served as positive and negative controls, respectively. Consistent with the described patterns for the gp140CFI_HXB2 Env glycoprotein expression (4), the gp140CFI_HxB2/89.6 mutant predominantly formed trimeric and dimeric oligomers (Fig. 1B, lane 3). Notably, there was a significant component of trimers that migrated as a well-resolved band with a molecular mass of 420 kDa, showing that the gp140CFI_HxB2/89.6 Env is capable of forming the appropriate oligomer. Typical of such analyses (66), some aggregation has also occurred based on the presence of additional high-molecular-mass (>500-kDa) multimers. Despite a similar pattern of oligomerization, the gp140CFI_HxB2/89.6 and gp140CFI_HXB2 glycoproteins could be distinguished from each other based on a lower electrophoretic mobility of the gp140CFI_HxB2/89.6 mutant due to the presence of two additional N-glycosylation sites, which increased its molecular mass by 5 kDa (67).

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Expression of the gp140CFIHxB2/89.6 Env glycoprotein and induction of neutralizing Ab responses. A, Schematic representation of the gp140CFIHxB2/89.6 expression construct. In the HIV-1 gp140CFIHxB2/89.6 chimeric vector, the region from residues 105 to 363 in gp140CFIHXB2 was replaced with the corresponding sequence from the HIV-189.6 Env glycoprotein (in gray). Abbreviations for the restriction enzyme sites NsiI and Bsu36I are denoted as N and B, respectively. B, Expression of gp140CFIHxB2/89.6 and gp140CFIHXB2 glycoproteins in culture supernatants from 293T transfectants. Control cells were transfected with the backbone plasmid (lane 1). The oligomeric states of secreted gp140CFIHXB2 and gp140CFIHxB2/89.6 glycoproteins were confirmed by BN-PAGE on 4–12% Bis-Tris NuPAGE gel (lanes 2 and 3, respectively). C, Generation of neutralizing Ab response by the gp140CFIHxB2/89.6 vaccine in rabbits. Sera were collected from three rabbits before immunization and after the prime/boost immunization with the gp140CFIHXB2 DNA construct and recombinant protein as described in Materials and Methods. Both preimmune and postimmune sera were tested at a 1/10 dilution for neutralizing activity against a panel of HIV-1 isolates. Neutralizing Ab titers were analyzed as previously described (54 ). SDs are indicated.

The neutralizing Ab responses elicited by the gp140CFI_HxB2/89.6 vaccine were examined against a panel of clade B HIV-1 isolates, including 89.6, SF162, HxB2, TORNO, QH0692, and SS1196, (70). The preimmune and immune rabbit sera were diluted 10-fold and analyzed for neutralizing activities in Env-pseudotyped virus neutralization and luciferase reporter cell assay. Fig. 1C shows that at the 1/10 serum dilution, the gp140CFI_HxB2/89.6 construct-immunized rabbit sera had a mean (± SD) neutralization activity of 78 ± 16% against the homologous 89.6 virus. Other HIV-1 isolates were neutralized less effectively with the activities ranging from 27.5 to 48%. Collectively, these results demonstrated that the gp140CFI_HxB2/89.6 vaccine is capable of inducing a neutralizing response to functional Env glycoproteins on homologous and, to a lesser extent, some heterologous HIV-1 isolates.

IL-21 augments gp140CFI_HxB2/89.6 vaccine-induced protection against challenge with vBD3 vaccinia virus

Using a challenge model with Env_89.6-expressing recombinant vBD3 vaccinia virus as a surrogate for HIV-1 infection (2), we examined the ability of the gp140CFI_HxB2/89.6 vaccine to induce protective cellular responses in mice. Because the vaccinia virus does not incorporate the Env glycoprotein in the virion but expresses it only in the infected cells, the observed protection is predominantly mediated by the vaccine-induced CTLs rather than Ab responses. Groups of BALB/c mice (n = 5) were injected with 10, 30, 100, or 300 µg of gp140CFI_HxB2/89.6 vector in both hind legs in the quadriceps muscle. The immunization was repeated every 3 wk, and the vaccinated mice were challenged with vBD3 or vSC8 virus 5 wk after the third immunization. The latter vaccinia virus expressing -galactosidase served as a specificity control. Because vaccinia virus replicates most efficiently in ovaries (71), ovaries were removed 5 days after the challenge and tested for viral titers on a monolayer of HuTK^–143 cell line. As shown in Fig. 2A, the vaccinia titers in ovaries of mice immunized with 10 or 30 µg of the gp140CFI_HxB2/89.6 construct and challenged with vBD3 were 10-fold lower than in vSC8-challenged mice or in animals immunized with the sham plasmid. The highest level of protection was detected in mice immunized with 100 or 300 µg of the gp140CFI_HxB2/89.6 plasmid (p < 0.05). In these mice, the viral load was 100-fold lower compared with the vSC8-challenged counterparts. Similar results were obtained by Chakrabarti et al. (4) using 100 µg of the gp140CFI_HXB2 plasmid DNA for induction of optimal Env-specific immune responses in mice.

View larger version (32K): [in this window] [in a new window]   FIGURE 2. Protection against challenge with vBD3 vaccinia virus following immunization with the gp140CFIHxB2/89.6 construct alone or in combination with a plasmid-encoded IL-21. A, BALB/c mice were immunized i.m. with DNA without an insert (sham vector) or a vector expressing gp140CFIHxB2/89.6 Env (10, 30, 100, or 300 µg/mouse). Five weeks after the third immunization, the mice were challenged i.p. with 2 x 107 PFU of vBD3 or vSC8. Five days later, the mice were sacrificed, ovaries removed, homogenized, and assayed for vaccinia virus titers by plating serial 10-fold dilutions on HuTK–143 cells, staining with crystal violet, and counting plaques at each dilution. B, BALB/c mice were immunized with the gp140CFIHxB2/89.6 construct alone or in combination with a plasmid-encoded IL-21 (10, 20, or 40 µg/mouse) before a challenge with vBD3 or vSC8 virus. The vaccinia titers are presented as the mean log10± SD (error bars) of PFU per ovaries of five mice per group. *, p < 0.05 and **, p < 0.01.

We next used the vBD3 challenge approach to determine the effect of IL-21 delivery relative to the DNA vaccination on the level and duration of gp140CFI_HxB2/89.6-induced immune responses. The plasmid-encoded IL-21 (20 µg/mouse) was injected either simultaneously with the gp140CFI_HxB2/89.6 construct (100 µg/mouse) or 5 and 15 days after specific vaccination. Two additional booster immunizations with the gp140CFI_HxB2/89.6 construct alone or in combination with IL-21 vector were conducted every 3 wk. The level of Env-specific immune responses in the immunized and IL-21 plasmid-treated mice were monitored every 2 mo by measuring protection against vBD3 challenge. To determine whether any protection was Env-specific, additional groups of the immunized mice were challenged with vSC8 vaccinia virus. Fig. 3 shows that administration of IL-21 vector enhanced to a varying degree the overall protection level against vBD3 challenge all mice immunized with the gp140CFI_HxB2/89.6 vaccine. Consistent with the notion that IL-21 exposure to an immune system that has been primed with a specific Ag may lead to the optimal augmentation of immune responses (40), administration of IL-21 vector 5 days after gp140CFI_HxB2/89.6 vaccination elicited the highest attenuation of vBD3 replication that lasted for 6 mo after the last booster immunization (Fig. 3). The protection against vBD3 in mice that received IL-21 together with the gp140CFI_HxB2/89.6 vaccine or 15 days after immunization lasted for 3 mo, and was higher compared with that elicited by immunization with the gp140CFI_HxB2/89.6 vector only (Fig. 3, A and B). The observed protection against challenge with vBD3 was specific as no inhibition of vSC8 replication was detected in the same groups of the immunized mice.

View larger version (18K): [in this window] [in a new window]   FIGURE 3. Effect of IL-21 delivery relative to the gp140CFIHxB2/89.6 DNA immunization on the level and duration of the Env vaccine-induced protection against challenge with vBD3 virus. A, BALB/c mice were immunized three times with the gp140CFIHxB2/89.6 construct alone or in combination with IL-21. The IL-21 plasmid was injected simultaneously with the Env vaccine (day 0), or 5 and 15 days later. The immunized mice were challenged with 2 x 107 PFU of vBD3 () and vSC8 () 1 mo after the third immunization. The viral titers in ovaries were analyzed 5 days after the challenge and tested for vaccinia titers on a monolayer of HuTK–143 cell line. B, The titers of vBD3 and vSC8 in ovaries of gp140CFIHxB2/89.6-immunized and IL-21-treated mice were analyzed 3 mo after vaccination. C, The titers of vBD3 and vSC8 in ovaries of gp140CFIHxB2/89.6-immunized and IL-21-treated mice were analyzed 6 mo after vaccination. The values are presented as the mean log10± SD (error bars) of PFU per ovaries of five mice per group. *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

IL-21 acts synergistically with IL-15 to enhance gp140CFI_HxB2/89.6-mediated protection

IL-21 has been reported to synergize with IL-15 in boosting Ag-specific CD8⁺ T cell expansion ex vivo, and during an adoptive transfer of B16 melanoma in sublethally irradiated mice (41). To further explore the actions of IL-21 and test its ability to synergize with IL-15 in enhancing DNA vaccine-induced immune responses, we examined to what extent IL-21 expression vector alters the immune response to the Env glycoprotein in gp140CFI_HxB2/89.6-immunized and IL-15-treated BALB/c mice. The optimal dose of the IL-15 expression vector (20 µg/mouse), determined by the vBD3 challenge study (data not shown), was administered together with the gp140CFI_HxB2/89.6 construct as previously described for the Env DNA vaccine (8), whereas IL-21 plasmid was delivered 5 days after the specific immunization. All mice were challenged i.p. with vBD3 or vSC8 vaccinia virus 5 wk after the third vaccination and analyzed for the protection level.

Consistent with results of the previous experiments, the vBD3 titer in ovaries of gp140CFI_HxB2/89.6-immunized mice was 100-fold lower compared with vSC8-challenged animals (Fig. 4A). Administration of IL-15 or IL-21 gene had a similar effect on the vaccine-induced protection reducing replication of vBD3 virus by 5 log₁₀ (p < 0.001). However, the combination of IL-15 and IL-21 plasmids was more potent than the effect of either cytokine alone on the gp140CFI_HxB2/89.6 vaccine-induced protection providing almost complete eradication of the virus. There were no significant differences between vSC8 titers in control mice and those immunized with the gp140CFI_HxB2/89.6 vaccine and treated with the cytokine genes, indicating that the observed synergy of IL-21 and IL-15 in providing protection against the vBD3 challenge could not be mediated by nonspecific inflammatory responses.

View larger version (22K): [in this window] [in a new window]   FIGURE 4. Effect of IL-15 and IL-21 genes on the Env vaccine-induced CD8+ T cell responses. A, Protection against vBD3 challenge. Five weeks after the third immunization with the gp140CFIHxB2/89.6 construct in the presence, absence or combination of IL-15 and IL-21 genes, BALB/c mice were challenged i.p. with 2 x 107 PFU of vBD3 () or vSC8 (). Control mice were immunized with the sham vector. Five days later, mice were sacrificed, ovaries removed, homogenized, and assayed for vaccinia virus titers. The titers are presented as the mean log10± SD (error bars) of PFU per ovaries of five mice per group. B, Analysis of Env-specific CTL responses in mice immunized with the gp140CFI89.6 construct in the presence, absence or combination of IL-15 and IL-21 expression vectors. The Env-specific CTL response was analyzed in a standard 51Cr release assay against P815 cells infected with vBD3 (solid symbols) or vSC8 (open symbols). All determinations were made in triplicate samples, and the SD was <10%. Results are presented as the means ± SD (error bars) of three independent experiments. C, The numbers of IFN--secreting CD8+ T cells in splenocyte cultures established from mice immunized with the gp140CFIHxB2/89.6 construct were determined by ELISPOT assay with vBD3-infected P815 cells. Control cells derived from mice immunized with the sham plasmid. Results are presented as the means ± SD (error bars) of at least three independent experiments. The frequencies of IFN--secreting cells were determined by regression analysis from a curve generated by plotting the number of spots vs the number of effector cells. *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

We next analyzed IL-15- and IL-21-induced changes in CD8⁺ T cells responses elicited by the gp140CFI_HxB2/89.6 vaccine in BALB/c mice by a standard 4-h ⁵¹Cr release assay against vBD3-infected P815 mastocytoma cells expressing only the MHC class I (H-2^d) molecules (59). Target cells infected with vSC8 virus or splenocytes from sham plasmid-immunized mice were used as specificity controls. Consistent with the in vivo analyses, the highest level of Env-specific CTL activities was measured in mice coimmunized with the IL-15 and IL-21 expression vectors (Fig. 4B). In these animals, vBD3-infected cells were lyzed with the highest level over a broad range of E:T ratios. The gp140CFI_HxB2/89.6 vaccine-induced CTL activities in mice that received either IL-21 or IL-15 vector were at relatively comparable levels and 2-fold higher than those in the untreated animals.

In parallel experiments, the effect of IL-15 and IL-21 genes on the frequency of IFN--secreting CD8⁺ splenocytes in gp140CFI_HxB2/89.6-immunized and control mice was analyzed by ELISPOT assay against vBD3-infected P815 cells. As shown in Fig. 4C, the IL-15 and IL-21 combination resulted in a marked increase in the number of IFN--secreting CD8⁺ T cells compared with that detected in gp140CFI_HxB2/89.6-immunized mice (p < 0.001). Mice that were treated with the plasmid-derived IL-15 at the time of specific vaccination also had elevated numbers of IFN--secreting CD8⁺ T cells, and higher than those elicited by the IL-21 treatment.

IL-15 and IL-21 enhance the expansion of CD8⁺ T cells to a subdominant Env_121–129 epitope in gp140CFI_HxB2/89.6-immunized HLA-A2/K^b mice

As new approaches for increasing the size and breadth of effector and memory pools induced after immunization are needed to improve the prospect of generating an effective HIV-1 vaccine, we investigated whether IL-15 and IL-21 cytokines would augment the vaccine-induced CD8⁺ T cells to the subdominant Env_121–129 epitope (KLTPLCVTL) with HLA-A*0201-binding motif. To this end, splenocytes from the HLA-A2/K^b-transgenic mice that were immunized with the gp140CFI_HxB2/89.6 vaccine in the presence or absence of IL-15 and IL-21 expression vectors were examined for Env_121–129/HLA-A*0201 tetramer⁺ CD8⁺ T cells by a flow cytometric analysis 5 wk after the third booster vaccination. As shown in Fig. 5, the frequency of tetramer-positive and CD8⁺ T cells in the immunized mice that were treated with IL-15 and IL-21 genes ranged from 2.1 x 10⁶ to 2.9 x 10⁶ cells, and were 2-fold higher compared with the untreated counterparts (2.43 ± 0.41 x 10⁶ cells vs 1.26 ± 0.22 x 10⁶ cells, p = 0.04). In the latter group of mice, the number of tetramer-positive CD8⁺ T cells ranged from 1.0 x 10⁶ to 1.5 x 10⁶ cells (Fig. 5A). To obtain a more comprehensive understanding of the effect of the plasmid-derived IL-15 and IL-21 on the memory cell development in gp140CFI_HxB2/89.6-immunized mice, we also analyzed the cell surface expression CD44, CD62L, and CD127 on Env_121–129/HLA-A*0201 tetramer⁺ CD8⁺ T cells. In both groups of mice, the majority of tetramer-positive CD8⁺ T cells expressed CD44 as well as CD62L and CD127 Ags (Fig. 5, A and B, right panels), suggesting that they could be selected to survive and develop into long-lived memory cells.

View larger version (26K): [in this window] [in a new window]   FIGURE 5. Expression of CD44, CD62L, and CD127 Ags on the cell surface of Env121–129/HLA-A*0201 tetramer+CD8+ splenocytes in the HLA-A2/Kb transgenic mice immunized with the gp140CFIHxB2/89.6 DNA vaccine alone or in combination with IL-15 and IL-21 genes. A, The HLA-A2/Kb-transgenic mice were immunized three times with the gp140CFIHxB2/89.6 DNA vaccine. The phenotypic analysis of splenocytes was performed on 5 wk after the last booster immunization. B, The gp140CFIHxB2/89.6-immunized HLA-A2/Kb-transgenic mice were injected with IL-15 and IL-21 genes as described in Materials and Methods. The phenotypic analysis of splenocytes was performed on 5 wk after the last booster immunization. Histograms depict vaccine-induced CD8+ T cells gated first for lymphocytes by forward scatter (FSC) and side scatter (SSC) and then for Env121–129 peptide-specific CD8+ T cells by tetramer and anti-CD8 mAb staining. Background staining was assessed using an isotype control Ab. The percent and mean fluorescent intensity (MFI) of CD44, CD62L, and CD127 molecules on Env121–129 peptide-specific CD8+ T cells are given. Arrows indicate the source of cells in the histograms. Data are from one representative experiment of three performed.

View larger version (30K): [in this window] [in a new window]   FIGURE 6. IL-21 acts synergistically with IL-51 in expanding the vaccine-induced CD8+ T cell responses to the subdominant Env121–129 epitope during vBD3 challenge. A, The phenotypic analysis of Env121–129 peptide-specific CD8+ T cells in splenocytes of the HLA-A2/Kb transgenic mice immunized with gp140CFIHxB2/89.6 DNA vaccine alone was performed on day 5 of vBD3 challenge. B, The effect of coimmunization with IL-15 and IL-21 genes on the expansion of the vaccine-induced CD8+ T cell responses to the subdominant Env121–129 epitope was analyzed 5 days after vBD3 challenge. Histograms depict vaccine-induced CD8+ T cells gated first for lymphocytes by forward scatter (FSC) and side scatter (SSC) and then for Env121–129 peptide-specific CD8+ T cells by tetramer and anti-CD8 mAb staining. Background staining was assessed using an isotype control Ab. The percent and MFI of CD44, CD62L, and CD127 on Env121–129 peptide-specific CD8+ T cells are given. Arrows indicate the source of cells in the histograms. The results are representative of four independent experiments.

To examine whether IL-15 and IL-21 gene delivery exerts a regulatory effect on the gp140CFI_HxB2/89.6 vaccine-induced T cell responses in vivo, we performed depletion experiments of the vaccine-induced CD8⁺ and CD4⁺ T cells during the vBD3 challenge study 5 wk after the third immunization. BALB/c mice that were immunized with the gp140CFI_HxB2/89.6 vaccine alone or in the presence of IL-15 and IL-21 plasmids were treated with anti-CD4 (clone GK 1.5) or anti-CD8 (clone 53–6.72) mAb. An additional group of the immunized mice was depleted of NK cells, and a control group was treated with rat IgG Ab. As shown in Table I, depletion of either CD4⁺ T cells or NK cells had no substantial effect on the vBD3 replication in ovaries of gp140CFI_HxB2/89.6-immunized mice as the difference in viral titers did not exceed 10% of that measured in animals treated with rat IgG Ab. In contrast, depletion of CD8⁺ T cells almost completely eliminated the vaccine-induced protection as the vBD3 titer in these mice was similar to that measured in animals treated with rat IgG or challenged with vSC8 virus. Similarly, depletion of CD8⁺ T cells in mice immunized with the gp140CFI_HxB2/89.6 construct in the presence of IL-15 and IL-21 plasmids almost completely eradicated Env-specific protective responses against vBD3. Table I shows that the vBD3 titer increased from 0.3 ± 0.2 log₁₀ PFU in the immunized mice treated with rat IgG to 7.3 ± 0.6 log₁₀ PFU in animals receiving CD8-specific mAb at the time of the challenge (p < 0.0001). Conversely, depletion of CD4⁺ T cells and NK cells had <25% effect on viral replication (Table I), indicating that the synergistic benefit provided by IL-15 and IL-21 treatment was largely dependent on the presence of Env-specific CD8⁺ T cells.

Based on the previous observation that coimmunization with a plasmid-delivered IL-15 results in enhanced function and longevity of CD8⁺ T cells that are partially independent of CD4⁺ T cell help (72), we tested whether IL-15 and IL-21 genes would enhance the vaccine-induced protection against vBD3 vaccinia virus challenge in mice that were partially depleted of CD4⁺ T cells. In these studies, BALB/c mice were injected i.p. with anti-CD4 mAb to deplete CD4⁺ T cells before vaccination with the gp140CFI_HxB2/89.6 plasmid. This treatment continued every 5 days during the entire period of three immunizations and until the challenge with vBD3 or vSC8 vaccinia virus performed 1 mo later. Additional groups of mice were depleted of CD8⁺ T cells by a similar treatment with anti-CD8 mAb. Analyses of the numbers of CD4⁺ and CD8⁺ T cells in the periphery by flow cytometry revealed that they were kept below 8% for the duration of the study. In BALB/c mice that maintained both CD4⁺ and CD8⁺ T cells during immunization with the gp140CFI_HxB2/89.6 construct only, the vBD3 titer in ovaries was 100-fold lower than that in vSC8-challenged animals (Fig. 7A; p < 0.05). In contrast, CD4 T cell-depleted mice that were immunized with the gp140CFI_HxB2/89.6 vaccine resulted in lack of protective immunity against vBD3 challenge, supporting the need for CD4⁺ Th cells during priming in the generation of functional CD8⁺ effector T cells. Also, the vaccine-primed and CD8⁺ T cell subset-depleted mice were unable to inhibit replication of the vBD3 vaccinia virus (Fig. 7A).

View larger version (17K): [in this window] [in a new window]   FIGURE 7. The generation of functional CD8+ T cells in gp140CFIHxB2/89.6-immunized mice requires CD4+ T cells during priming and is partially restored by coimmunization with IL-15 and IL-21 genes in a model of CD4+ Th cell depletion. A, BALB/c mice were depleted of CD4+ T cells before vaccination with the gp140CFIHxB2/89.6 construct by i.p. injection of anti-CD4 mAb. Additional groups of mice were depleted of CD8+ T cells by treatment with anti-CD8 mAb. The Ab treatment continued every 5 days during the entire period of three immunizations and until the challenge studies with vBD3 () and vSC8 () vaccinia viruses performed 1 mo later. B, BALB/c mice were depleted of CD4+ T cells before vaccination with the gp140CFIHxB2/89.6 construct and treatment with the combination of IL-15 and IL-21genes by i.p. injection of anti-CD4 mAb. In all experiments, IL-15 was delivered together with the vaccine, whereas IL-21 expression vector was injected 5 days later. Additional groups of mice were depleted of CD8+ T cells by treatment with anti-CD8 mAb. The vaccinia titers are presented as the mean log10± SD (error bars) of PFU per ovaries of four mice per group. *, p < 0.05; ***, p < 0.0001.

IL-15 and IL-21 enhance humoral responses and the effector function of Env-specific Abs in gp140CFI_HxB2/89.6-immunized mice

Based on the previous studies which demonstrated that IL-21 regulates humoral immunity by promoting B cell maturation during a productive T lymphocyte-dependent B cell response while favoring growth arrest and apoptosis for nonspecifically or inappropriately activated B cells (73, 74), we sought to examine the effect of IL-21 expression vector delivered alone or in combination with IL-15 gene on the gp140CFI_HxB2/89.6 vaccine-induced Ab responses. BALB/c mice were immunized three times with the gp140CFI_HxB2/89.6 construct in the presence, absence or combination of IL-15 and IL-21 genes. In all experiments, IL-15 was delivered together with the vaccine, whereas IL-21 expression vector was injected 5 days later. The titers of Env-specific IgM Abs in sera of gp140CFI_89.6-immunized mice were analyzed after each immunization. The ELISA with plates coated with the Env glycoprotein was used to measure the end-point titers and isotypes of the vaccine-induced serum Abs collected 3 wk after each booster immunization. As shown in Fig. 8A, detectable levels of Env-specific IgM Ab responses were observed already after the first immunization with the gp140CFI_HxB2/89.6 vector with only small differences among mice vaccinated in the presence or absence of IL-15 and IL-21 plasmids. The titers of IgM Abs in gp140CFI_HxB2/89.6-immunized mice increased after the second immunization in all groups of the immunized mice (p < 0.05), but showed only moderate enhancement after the third immunization. Moreover, similar anti-Env IgM Ab responses were observed in all immunization groups after each booster vaccination, suggesting that IL-15 and IL-21 administered either alone or in combination had no substantial effect on the gp140CFI_HxB2/89.6-induced IgM Ab level. In contrast, coimmunization with IL-15 and IL-21 expression vectors augmented Env-specific IgG Ab responses, which were most prominent after the second and third booster immunization with the gp140CFI_HxB2/89.6 vaccine (Fig. 8B). We also observed that IL-21 preferentially augmented Env-specific IgG1 Abs and had a modest effect on gp140CFI_HxB2/89.6-induced IgG2, whereas IL-15 plasmid increased Env-specific IgG2a as well as IgG2b Ab production (Fig. 8C). None of the cytokines affected production of Env-specific IgG3 Ab responses, which remained at a low level in all groups of the immunized mice.

View larger version (29K): [in this window] [in a new window]   FIGURE 8. Env-specific Ab responses induced by vaccination of BALB/c mice with the gp140CFI89.6 construct in the presence, absence, or combination of IL-15 and IL-21 genes. A, BALB/c mice were immunized three times with the gp140CFIHxB2/89.6 construct in the presence, absence, or combination of IL-15 and IL-21 genes. In all experiments, IL-15 was delivered together with the vaccine, whereas IL-21 expression vector was injected 5 days later. The titers of Env-specific IgM Abs in sera of gp140CFI89.6-immunized mice were determined after each immunization by ELISA using alkaline phosphatase-conjugated secondary Abs. B, The titers of Env-specific IgG Abs were analyzed in the same sera of the gp140CFI89.6 construct-immunized mice. C, The titers of Env-specific IgG subclasses were determined in sera after the third immunization with the gp140CFI89.6 construct in the presence, absence, or combination of IL-15 and IL-21 genes. Results are presented as the means ± SD (error bars) of three independent experiments. *, p < 0.05.

View larger version (17K): [in this window] [in a new window]   FIGURE 9. ADCC and CDC against Env-expressing cells mediated by sera of mice immunized with the gp140CFI89.6 construct in the presence, absence or combination of IL-15 and IL-21 genes. A, ADCC was measured using vBD3-infected (solid symbols) and vSC8-infected (open symbols) target cells and serial dilutions of sera collected 3 wk after the third immunization with the Env vaccine in the presence, absence, or combination of IL-15 and IL-21 genes. B, The complement-mediated lysis of vBD3-infected (black symbols) and vSC8-infected (white symbols) target cells was determined using the same sera as for the ADCC assay. All determinations were made in triplicate samples, and the SD was <10%. Results are presented as the means ± SD (error bars) of three independent experiments.

	Discussion

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The current paradigms hold that memory CD8⁺ T cells represent highly fit cells derived from the effector T cell population (79, 80, 81), thus leading to the prediction that therapies which augment T cell effector pools may also augment T cell memory pools (82, 83). Our results are consistent with this hypothesis as well as the most recent studies which show that rIL-21 cooperates with IL-15 in achieving the expansion of tumor-specific CD8⁺ T cells associated with regression of B16 melanoma (41). The present study further extends these observations and demonstrates for the first time a synergistic action of IL-15 and IL-21 genes in augmenting the efficacy of HIV-1 Env-specific DNA vaccine through induction of durable Env-specific immune responses which were protective against vBD3 vaccinia virus challenge.

Similar to the results obtained by Zeng et al. (41), we found that the effect of IL-21 gene on the vaccine-induced immune responses is dose dependent and optimal at 20 µg of plasmid DNA/mouse. Interestingly, injection of the IL-21-encoded plasmid 5 days after specific vaccination induced the highest and long-lasting protection, whereas delivery of the plasmid simultaneous with specific vaccination produced suboptimal benefit for the enhancing of specific immunity. These findings are consistent with previous reports in which IL-21 addition led to an optimal augmentation of the specific T cell responses when provided after Ag priming (40, 84). They are also in an agreement with the recent data indicating that IL-21 expression during challenge with HSV-2 or lymphocytic choriomeningitis virus is elevated on days 5 and 7 and coincide with the onset of the adaptive immune response (85). Altogether, these studies suggest that IL-21 promotes adaptive immunity by regulating the expansion and/or restoration of vaccine-induced T cell responses, and only marginally affects Ag priming of naive cells. This possibility is also supported by another study which demonstrated that IL-21 protein used at concentrations of 20 and 100 ng/ml blocked the LPS-induced maturation of dendritic cells in vitro (86). Similarly, we found that administration of recombinant mouse IL-21 protein (>10 µg per injection) at the time of the DNA vaccination and for 7 consecutive days reduced the level of Env-specific T cell responses and protection (data not shown), suggesting that when delivered at suboptimal concentrations before vaccines, IL-21 may diminish Ag presentation. These results together with those of previous studies which showed that IL-15 regulates naive T cell viability and promotes the maintenance of memory T cells (18), suggest that a dual approach of expanding the pool of activated CD8⁺ T cells with IL-15 together with promoting their differentiation with IL-21 may augment the vaccine-specific immune responses.

Another major impediment to the development of successful vaccines against HIV-1, which requires broadly reactive cellular immune responses for overcoming mutations to target Ags as well as anergy and/or suppression due to chronic Ag overload, is the limited number of vaccine modalities and adjuvants capable of eliciting CD4⁺ and CD8⁺ T cell responses. It is believed that CD8⁺ T cell responses are important for controlling HIV-1 infection and slowing disease progression. Although the exact function of HIV-1-specific CD8⁺ T cells in this process has not been fully elucidated, a correlation has been established between long-term nonprogression of HIV-1 in seropositive individuals and HIV-1-specific CD8⁺ T cells to maintain effector T cell activity and control virus. This suggests that understanding the factors that supplement CD4⁺ T cell function and support CD8⁺ T cell activity could be important for HIV-1 therapy strategy where CD4 help is compromised. Therefore, the reported ability of IL-15 plasmid to restore CD8⁺ T cell secondary immune responses to an antigenic DNA plasmid in the partial absence of CD4⁺ T cells may be useful in contributing to vaccine control of HIV-1 infection (72). Our results that the combination of IL-15 and IL-21 enhanced the vaccine-induced protection against vBD3 vaccinia virus challenge in mice that were partially depleted of CD4⁺ T cells are supportive of the previous findings. It has been suggested that IL-15 directly activates CD8⁺ T cells during the priming stage, leading to increased formation of memory cells (72). Alternatively, IL-15 may directly activate APCs, leading to the secretion of IL-12, which subsequently induces IFN- to further activate dendritic cells and macrophages and provide costimulation of CD8⁺ T cells (72). Although these possibilities remain to be examined, we hypothesize that IL-21 may promote differentiation of IL-15-activated CD8⁺ T cells. In support of this possibility are our preliminary findings in the CD4 knockout mice which showed that coimmunization with IL-15 and IL-21 genes provides higher protection than IL-15 alone, whereas IL-21 vector had no substantial effect on the responses. Additional experiments are required to determine the mechanism by which the interaction between IL-15 and IL-21 cytokines would promote beneficial immune responses, especially in a model of CD4 depletion.

To design an effective vaccine, it is important to understand the types of immune responses required to prevent infection or disease progression. Many currently licensed vaccines mediate protection primarily by humoral immune responses, in which Abs bind and neutralize the pathogen. However, for many diseases including AIDS, it can be envisioned to be difficult for vaccines to elicit broadly neutralizing Abs. In such cases, adaptive cellular immune responses together with innate immunity are critical for control of the pathogen and protection against disease progression, although both humoral and cellular immune responses will likely be required for optimal and sustained protection. Consistent with the reported results in a B16 melanoma model (41), our data demonstrate that both IL-15 and IL-21 synergize in promoting the generation and survival of gp140CFI_HxB2/89.6 vaccine-induced durable cellular responses capable of eradicating vBD3 infection in association with the expansion of Env-specific CD8⁺ T cells. Moreover, we also found that combination of IL-15 and IL-21 plasmids increases the cytolytic effector function of humoral responses including ADCC and CDC through augmentation of Env-specific IgG Ab level. This, together with the highlighted importance of IL-21 in inducing long-lasting and effective CD8⁺ T responses (48, 87, 88, 89), suggests that IL-21-mediated immunomodulation warrants further evaluation for HIV-1 vaccine.

	Acknowledgments

 
We are grateful to Drs. Gary Nabel and Bernard Moss for the reagents. We thank Michelle Detwiler for DNA sequencing, Zane Kraft for performing the neutralization experiments, and Earl Timm for help with a flow cytometric analysis.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Research Grants R21 AI060375 and R21 AI54163 from the National Institute of Allergy and Infectious Diseases (to D.K.), funds to commemorate Dr. Goro Chihara’s research activity, grants from the National Institute of Allergy and Infectious Diseases R01AI47708 and R01 AI51217, and National Cancer Institute Grant CA104645 (to P.A.S.).

² Current address: Department of Biochemistry and Molecular Biology, University of Medical Sciences, Poznan, Poland.

³ Address correspondence and reprint requests to Dr. Danuta Kozbor, Department of Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. E-mail address: danuta.kozbor{at}roswellpark.org

⁴ Abbreviations used in this paper: _c, common receptor chain; ADCC, Ab-dependent cellular cytotoxicity; CDC, complement-dependent cytotoxicity; Env, envelope; RLU, relative light unit; rVV, recombinant vaccinia virus.

Received for publication May 19, 2005. Accepted for publication April 19, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Seaman, M. S., L. Xu, K. Beaudry, K. L. Martin, M. H. Beddall, A. Miura, A. Sambor, B. K. Chakrabarti, Y. Huang, R. Bailer, et al 2005. Multiclade human immunodeficiency virus type 1 envelope immunogens elicit broad cellular and humoral immunity in rhesus monkeys. J. Virol. 79: 2956-2963. [Abstract/Free Full Text]
2. Kiszka, I., D. Kmieciak, J. Gzyl, T. Naito, E. Bolesta, A. Sieron, S. P. Singh, A. Srinivasan, G. Trinchieri, Y. Kaneko, D. Kozbor. 2002. Effect of the V3 loop deletion of envelope glycoprotein on cellular responses and protection against challenge with recombinant vaccinia virus expressing gp160 of primary human immunodeficiency virus type 1 isolates. J. Virol. 76: 4222-4232. [Abstract/Free Full Text]
3. Donnelly, J. J., B. Wahren, M. A. Liu. 2005. DNA vaccines: progress and challenges. J. Immunol. 175: 633-639. [Abstract/Free Full Text]
4. Chakrabarti, B. K., W. P. Kong, B. Y. Wu, Z. Y. Yang, J. Friborg, X. Ling, S. R. King, D. C. Montefiori, G. J. Nabel. 2002. Modifications of the human immunodeficiency virus envelope glycoprotein enhance immunogenicity for genetic immunization. J. Virol. 76: 5357-5368. [Abstract/Free Full Text]
5. Hanke, T., A. J. McMichael, R. V. Samuel, L. A. Powell, L. McLoughlin, S. J. Crome, A. Edlin. 2002. Lack of toxicity and persistence in the mouse associated with administration of candidate DNA- and modified vaccinia virus Ankara (MVA)-based HIV vaccines for Kenya. Vaccine 21: 108-114. [Medline]
6. Belyakov, I. M., B. Moss, W. Strober, J. A. Berzofsky. 1999. Mucosal vaccination overcomes the barrier to recombinant vaccinia immunization caused by preexisting poxvirus immunity. Proc. Natl. Acad. Sci. USA 96: 4512-4517. [Abstract/Free Full Text]
7. Cayabyab, M. J., A. H. Hovav, T. Hsu, G. R. Krivulka, M. A. Lifton, D. A. Gorgone, G. J. Fennelly, B. F. Haynes, W. R. Jacobs, Jr, N. L. Letvin. 2006. Generation of CD8⁺ T-cell responses by a recombinant nonpathogenic Mycobacterium smegmatis vaccine vector expressing human immunodeficiency virus type 1 Env. J. Virol. 80: 1645-1652. [Abstract/Free Full Text]
8. Moore, A. C., W. P. Kong, B. K. Chakrabarti, G. J. Nabel. 2002. Effects of antigen and genetic adjuvants on immune responses to human immunodeficiency virus DNA vaccines in mice. J. Virol. 76: 243-250. [Abstract/Free Full Text]
9. Barouch, D. H., S. Santra, T. D. Steenbeke, X. X. Zheng, H. C. Perry, M. E. Davies, D. C. Freed, A. Craiu, T. B. Strom, J. W. Shiver, N. L. Letvin. 1998. Augmentation and suppression of immune responses to an HIV-1 DNA vaccine by plasmid cytokine/Ig administration. J. Immunol. 161: 1875-1882. [Abstract/Free Full Text]
10. Barouch, D. H., A. Craiu, M. J. Kuroda, J. E. Schmitz, X. X. Zheng, S. Santra, J. D. Frost, G. R. Krivulka, M. A. Lifton, C. L. Crabbs, et al 2000. Augmentation of immune responses to HIV-1 and simian immunodeficiency virus DNA vaccines by IL-2/Ig plasmid administration in rhesus monkeys. Proc. Natl. Acad. Sci. USA 97: 4192-4197. [Abstract/Free Full Text]
11. Chattergoon, M. A., V. Saulino, J. P. Shames, J. Stein, L. J. Montaner, D. B. Weiner. 2004. Co-immunization with plasmid IL-12 generates a strong T-cell memory response in mice. Vaccine 22: 1744-1750. [Medline]
12. Agadjanyan, M. G., M. A. Chattergoon, M. J. Holterman, B. Monzavi-Karbassi, J. J. Kim, T. Dentchev, D. Wilson, V. Ayyavoo, L. J. Montaner, T. Kieber-Emmons, et al 2003. Costimulatory molecule immune enhancement in a plasmid vaccine model is regulated in part through the Ig constant-like domain of CD80/86. J. Immunol. 171: 4311-4319. [Abstract/Free Full Text]
13. Xin, K. Q., K. Hamajima, S. Sasaki, T. Tsuji, S. Watabe, E. Okada, K. Okuda. 1999. IL-15 expression plasmid enhances cell-mediated immunity induced by an HIV-1 DNA vaccine. Vaccine 17: 858-866. [Medline]
14. Kim, J. J., K. A. Simbiri, J. I. Sin, K. Dang, J. Oh, T. Dentchev, D. Lee, L. K. Nottingham, A. A. Chalian, D. McCallus, et al 1999. Cytokine molecular adjuvants modulate immune responses induced by DNA vaccine constructs for HIV-1 and SIV. J. Interferon Cytokine Res. 19: 77-84. [Medline]
15. Pasquini, S., Z. Xiang, Y. Wang, Z. He, H. Deng, M. Blaszczyk-Thurin, H. C. Ertl. 1997. Cytokines and costimulatory molecules as genetic adjuvants. Immunol. Cell Biol. 75: 397-401. [Medline]
16. Min, W., H. S. Lillehoj, J. Burnside, K. C. Weining, P. Staeheli, J. J. Zhu. 2001. Adjuvant effects of IL-1, IL-2, IL-8, IL-15, IFN-, IFN- TGF-4 and lymphotactin on DNA vaccination against Eimeria acervulina. Vaccine 20: 267-274. [Medline]
17. Tagaya, Y., R. N. Bamford, A. P. DeFilippis, T. A. Waldmann. 1996. IL-15: a pleiotropic cytokine with diverse receptor/signaling pathways whose expression is controlled at multiple levels. Immunity 4: 329-336. [Medline]
18. Schluns, K. S., L. Lefrancois. 2003. Cytokine control of memory T-cell development and survival. Nat. Rev. Immunol. 3: 269-279. [Medline]
19. Waldmann, T., Y. Tagaya, R. Bamford. 1998. Interleukin-2, interleukin-15, and their receptors. Int. Rev. Immunol. 16: 205-226. [Medline]
20. D’Souza, W. N., K. S. Schluns, D. Masopust, L. Lefrancois. 2002. Essential role for IL-2 in the regulation of antiviral extralymphoid CD8 T cell responses. J. Immunol. 168: 5566-5572. [Abstract/Free Full Text]
21. Schluns, K. S., K. Williams, A. Ma, X. X. Zheng, L. Lefrancois. 2002. Cutting edge: requirement for IL-15 in the generation of primary and memory antigen-specific CD8 T cells. J. Immunol. 168: 4827-4831. [Abstract/Free Full Text]
22. Schluns, K. S., W. C. Kieper, S. C. Jameson, L. Lefrancois. 2000. Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo. Nat. Immunol. 1: 426-432. [Medline]
23. Lefrancois, L., D. Masopust. 2002. T cell immunity in lymphoid and non-lymphoid tissues. Curr. Opin. Immunol. 14: 503-508. [Medline]
24. Sprent, J., A. D. Judge, X. Zhang. 2002. Cytokines and memory-phenotype CD8⁺ cells. Adv. Exp. Med. Biol. 512: 147-153. [Medline]
25. Refaeli, Y., L. Van Parijs, C. A. London, J. Tschopp, A. K. Abbas. 1998. Biochemical mechanisms of IL-2-regulated Fas-mediated T cell apoptosis. Immunity 8: 615-623. [Medline]
26. Tanchot, C., F. A. Lemonnier, B. Perarnau, A. A. Freitas, B. Rocha. 1997. Differential requirements for survival and proliferation of CD8 naive or memory T cells. Science 276: 2057-2062. [Abstract/Free Full Text]
27. Tough, D. F., J. Sprent. 1995. Life span of naive and memory T cells. Stem Cells 13: 242-249. [Medline]
28. Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh, R. Ahmed. 2003. Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 4: 1191-1198. [Medline]
29. Kanai, T., E. K. Thomas, Y. Yasutomi, N. L. Letvin. 1996. IL-15 stimulates the expansion of AIDS virus-specific CTL. J. Immunol. 157: 3681-3687. [Abstract]
30. Ku, C. C., M. Murakami, A. Sakamoto, J. Kappler, P. Marrack. 2000. Control of homeostasis of CD8⁺ memory T cells by opposing cytokines. Science 288: 675-678. [Abstract/Free Full Text]
31. Judge, A. D., X. Zhang, H. Fujii, C. D. Surh, J. Sprent. 2002. Interleukin 15 controls both proliferation and survival of a subset of memory-phenotype CD8⁺ T cells. J. Exp. Med. 196: 935-946. [Abstract/Free Full Text]
32. Becker, T. C., E. J. Wherry, D. Boone, K. Murali-Krishna, R. Antia, A. Ma, R. Ahmed. 2002. Interleukin 15 is required for proliferative renewal of virus-specific memory CD8 T cells. J. Exp. Med. 195: 1541-1548. [Abstract/Free Full Text]
33. Vella, A. T., S. Dow, T. A. Potter, J. Kappler, P. Marrack. 1998. Cytokine-induced survival of activated T cells in vitro and in vivo. Proc. Natl. Acad. Sci. USA 95: 3810-3815. [Abstract/Free Full Text]
34. Armitage, R. J., B. M. Macduff, J. Eisenman, R. Paxton, K. H. Grabstein. 1995. IL-15 has stimulatory activity for the induction of B cell proliferation and differentiation. J. Immunol. 154: 483-490. [Abstract]
35. Oh, S., J. A. Berzofsky, D. S. Burke, T. A. Waldmann, L. P. Perera. 2003. Coadministration of HIV vaccine vectors with vaccinia viruses expressing IL-15 but not IL-2 induces long-lasting cellular immunity. Proc. Natl. Acad. Sci. USA 100: 3392-3397. [Abstract/Free Full Text]
36. Araki, A., S. Hazama, K. Yoshimura, S. Yoshino, N. Iizuka, M. Oka. 2004. Tumor secreting high levels of IL-15 induces specific immunity to low immunogenic colon adenocarcinoma via CD8⁺ T cells. Int. J. Mol. Med. 14: 571-576. [Medline]
37. Villinger, F., R. Miller, K. Mori, A. E. Mayne, P. Bostik, J. B. Sundstrom, C. Sugimoto, A. A. Ansari. 2004. IL-15 is superior to IL-2 in the generation of long-lived antigen specific memory CD4 and CD8 T cells in rhesus macaques. Vaccine 22: 3510-3521. [Medline]
38. Umemura, M., H. Nishimura, K. Saito, T. Yajima, G. Matsuzaki, S. Mizuno, I. Sugawara, Y. Yoshikai. 2003. Interleukin-15 as an immune adjuvant to increase the efficacy of Mycobacterium bovis bacillus Calmette-Guerin vaccination. Infect. Immun. 71: 6045-6048. [Abstract/Free Full Text]
39. Rubinstein, M. P., A. N. Kadima, M. L. Salem, C. L. Nguyen, W. E. Gillanders, D. J. Cole. 2002. Systemic administration of IL-15 augments the antigen-specific primary CD8⁺ T cell response following vaccination with peptide-pulsed dendritic cells. J. Immunol. 169: 4928-4935. [Abstract/Free Full Text]
40. Moroz, A., C. Eppolito, Q. Li, J. Tao, C. H. Clegg, P. A. Shrikant. 2004. IL-21 enhances and sustains CD8⁺T cell responses to achieve durable tumor immunity: comparative evaluation of IL-2, IL-15, and IL-21. J. Immunol. 173: 900-909. [Abstract/Free Full Text]
41. Zeng, R., R. Spolski, S. E. Finkelstein, S. Oh, P. E. Kovanen, C. S. Hinrichs, C. A. Pise-Masison, M. F. Radonovich, J. N. Brady, N. P. Restifo, et al 2005. Synergy of IL-21 and IL-15 in regulating CD8⁺ T cell expansion and function. J. Exp. Med. 201: 139-148. [Abstract/Free Full Text]
42. van Leeuwen, E. M., L. E. Gamadia, P. A. Baars, E. B. Remmerswaal, I. J. ten Berge, R. A. van Lier. 2002. Proliferation requirements of cytomegalovirus-specific, effector-type human CD8⁺ T cells. J. Immunol. 169: 5838-5843. [Abstract/Free Full Text]
43. Kasaian, M. T., M. J. Whitters, L. L. Carter, L. D. Lowe, J. M. Jussif, B. Deng, K. A. Johnson, J. S. Witek, M. Senices, R. F. Konz, et al 2002. IL-21 limits NK cell responses and promotes antigen-specific T cell activation: a mediator of the transition from innate to adaptive immunity. Immunity 16: 559-569. [Medline]
44. Ozaki, K., R. Spolski, C. G. Feng, C. F. Qi, J. Cheng, A. Sher, H. C. Morse, III, C. Liu, P. L. Schwartzberg, W. J. Leonard. 2002. A critical role for IL-21 in regulating immunoglobulin production. Science 298: 1630-1634. [Abstract/Free Full Text]
45. Parrish-Novak, J., S. R. Dillon, A. Nelson, A. Hammond, C. Sprecher, J. A. Gross, J. Johnston, K. Madden, W. Xu, J. West, et al 2000. Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function. Nature 408: 57-63. [Medline]
46. Suto, A., H. Nakajima, K. Hirose, K. Suzuki, S. Kagami, Y. Seto, A. Hoshimoto, Y. Saito, D. C. Foster, I. Iwamoto. 2002. Interleukin 21 prevents antigen-induced IgE production by inhibiting germ line C() transcription of IL-4-stimulated B cells. Blood 100: 4565-4573. [Abstract/Free Full Text]
47. Cui, F. D., H. Asada, M. L. Jin, T. Kishida, M. Shin-Ya, T. Nakaya, M. Kita, M. Ishii, M. Iwai, T. Okanoue, et al 2005. Cytokine genetic adjuvant facilitates prophylactic intravascular DNA vaccine against acute and latent herpes simplex virus infection in mice. Gene Ther. 12: 160-168. [Medline]
48. Kishida, T., H. Asada, Y. Itokawa, F. D. Cui, M. Shin-Ya, S. Gojo, K. Yasutomi, Y. Ueda, H. Yamagishi, J. Imanishi, O. Mazda. 2003. Interleukin (IL)-21 and IL-15 genetic transfer synergistically augments therapeutic antitumor immunity and promotes regression of metastatic lymphoma. Mol. Ther. 8: 552-558. [Medline]
49. Wang, G., M. Tschoi, R. Spolski, L. Y., K. Ozaki, C. Feng, G. Kim, W. J. Leonard, and P. Hwu. 2003. In vivo antitumor activity of interleukin 21 mediated by natural killer cells. Cancer Res. 63: 9016–9022.
50. Comes, A., O. Rosso, A. M. Orengo, E. Di Carlo, C. Sorrentino, R. Meazza, T. Piazza, B. Valzasina, P. Nanni, M. P. Colombo, S. Ferrini. 2006. CD25⁺ regulatory T cell depletion augments immunotherapy of micrometastases by an IL-21-secreting cellular vaccine. J. Immunol. 176: 1750-1758. [Abstract/Free Full Text]
51. Kmieciak, D., T. J. Wasik, H. Teppler, J. Pientka, S. H. Hsu, H. Takahashi, K. Okumura, Y. Kaneko, D. Kozbor. 1998. The effect of deletion of the V3 loop of gp120 on cytotoxic T cell responses and HIV gp120-mediated pathogenesis. J. Immunol. 160: 5676-5683. [Abstract/Free Full Text]
52. Schulke, N., M. S. Vesanen, R. W. Sanders, P. Zhu, M. Lu, D. J. Anselma, A. R. Villa, P. W. Parren, J. M. Binley, K. H. Roux, et al 2002. Oligomeric and conformational properties of a proteolytically mature, disulfide-stabilized human immunodeficiency virus type 1 gp140 envelope glycoprotein. J. Virol. 76: 7760-7776. [Abstract/Free Full Text]
53. Mascola, J. R., M. K. Louder, C. Winter, R. Prabhakara, S. C. De Rosa, D. C. Douek, B. J. Hill, D. Gabuzda, M. Roederer. 2002. Human immunodeficiency virus type 1 neutralization measured by flow cytometric quantitation of single-round infection of primary human T cells. J. Virol. 76: 4810-4821. [Abstract/Free Full Text]
54. Saunders, C. J., R. A. McCaffrey, I. Zharkikh, Z. Kraft, S. E. Malenbaum, B. Burke, C. Cheng-Mayer, L. Stamatatos. 2005. The V1, V2, and V3 regions of the human immunodeficiency virus type 1 envelope differentially affect the viral phenotype in an isolate-dependent manner. J. Virol. 79: 9069-9080. [Abstract/Free Full Text]
55. Doranz, B. J., J. Rucker, Y. Yi, R. J. Smyth, M. Samson, S. C. Peiper, M. Parmentier, R. G. Collman, R. W. Doms. 1996. A dual-tropic primary HIV-1 isolate that uses fusin and the -chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell 85: 1149-1158. [Medline]
56. Earl, P. L., S. Koenig, B. Moss. 1991. Biological and immunological properties of human immunodeficiency virus type 1 envelope glycoprotein: analysis of proteins with truncations and deletions expressed by recombinant vaccinia viruses. J. Virol. 65: 31-41. [Abstract/Free Full Text]
57. Binder, D., T. M. Kundig. 1991. Antiviral protection by CD8⁺ versus CD4⁺ T cells: CD8⁺ T cells correlating with cytotoxic activity in vitro are more efficient in antivaccinia virus protection than CD4-dependent IL. J. Immunol. 146: 4301-4307. [Abstract]
58. Okuda, K., A. Ihata, S. Watabe, E. Okada, T. Yamakawa, K. Hamajima, J. Yang, N. Ishii, M. Nakazawa, K. Ohnari, et al 2001. Protective immunity against influenza A virus induced by immunization with DNA plasmid containing influenza M gene. Vaccine 19: 3681-3691. [Medline]
59. Gherardi, M. M., M. Esteban. 1999. Mucosal and systemic immune responses induced after oral delivery of vaccinia virus recombinants. Vaccine 17: 1074-1083. [Medline]
60. Ueno, T., H. Tomiyama, M. Takiguchi. 2002. Single T cell receptor-mediated recognition of an identical HIV-derived peptide presented by multiple HLA class I molecules. J. Immunol. 169: 4961-4969. [Abstract/Free Full Text]
61. Staats, H. F., W. G. Nichols, T. J. Palker. 1996. Mucosal immunity to HIV-1: systemic and vaginal antibody responses after intranasal immunization with the HIV-1 C4/V3 peptide T1SP10 MN(A). J. Immunol. 157: 462-472. [Abstract]
62. Nishimura, T., T. Itoh. 1988. Higher level expression of lymphocyte function-associated antigen-1 (LFA-1) on in vivo natural killer cells. Eur. J. Immunol. 18: 2077-2080. [Medline]
63. Yanagita, Y., T. Nishimura, X. H. Gao, T. Mitomi, S. Habu. 1992. Natural cytotoxic T cells responsible for anti-CD3-induced cytotoxicity in mice. Immunol. Lett. 31: 137-142. [Medline]
64. Batova, A., A. Kamps, S. D. Gillies, R. A. Reisfeld, A. L. Yu. 1999. The Ch14.18-GM-CSF fusion protein is effective at mediating antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in vitro. Clin. Cancer Res. 5: 4259-4263. [Abstract/Free Full Text]
65. Winer, B. J.. 1971. Design and analysis of factorial experiments. Statistical Principles in Experimental Design 224-251. McGraw-Hill, New York.
66. Yang, X., M. Farzan, R. Wyatt, J. Sodroski. 2000. Characterization of stable, soluble trimers containing complete ectodomains of human immunodeficiency virus type 1 envelope glycoproteins. J. Virol. 74: 5716-5725. [Abstract/Free Full Text]
67. Kornfeld, R., S. Kornfeld. 1985. Assembly of asparagine-linked oligosaccharides. Annu. Rev. Biochem. 54: 631-664. [Medline]
68. Burton, D. R., R. C. Desrosiers, R. W. Doms, W. C. Koff, P. D. Kwong, J. P. Moore, G. J. Nabel, J. Sodroski, I. A. Wilson, R. T. Wyatt. 2004. HIV vaccine design and the neutralizing antibody problem. Nat. Immunol. 5: 233-236. [Medline]
69. Gzyl, J., E. Bolesta, A. Wierzbicki, D. Kmieciak, T. Naito, M. Honda, K. Komuro, Y. Kaneko, D. Kozbor. 2004. Effect of partial and complete variable loop deletions of the human immunodeficiency virus type 1 envelope glycoprotein on the breadth of gp160-specific immune responses. Virology 318: 493-506. [Medline]
70. Li, M., F. Gao, J. R. Mascola, L. Stamatatos, V. R. Polonis, M. Koutsoukos, G. Voss, P. Goepfert, P. Gilbert, K. M. Greene, et al 2005. Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies. J. Virol. 79: 10108-10125. [Abstract/Free Full Text]
71. Belyakov, I. M., M. A. Derby, J. D. Ahlers, B. L. Kelsall, P. Earl, B. Moss, W. Strober, J. A. Berzofsky. 1998. Mucosal immunization with HIV-1 peptide vaccine induces mucosal and systemic cytotoxic T lymphocytes and protective immunity in mice against intrarectal recombinant HIV-vaccinia challenge. Proc. Natl. Acad. Sci. USA 95: 1709-1714. [Abstract/Free Full Text]
72. Kutzler, M. A., T. M. Robinson, M. A. Chattergoon, D. K. Choo, A. Y. Choo, P. Y. Choe, M. P. Ramanathan, R. Parkinson, S. Kudchodkar, Y. Tamura, et al 2005. Coimmunization with an optimized IL-15 plasmid results in enhanced function and longevity of CD8 T cells that are partially independent of CD4 T cell help. J. Immunol. 175: 112-123. [Abstract/Free Full Text]
73. Mehta, D. S., A. L. Wurster, M. J. Whitters, D. A. Young, M. Collins, M. J. Grusby. 2003. IL-21 induces the apoptosis of resting and activated primary B cells. J. Immunol. 170: 4111-4118. [Abstract/Free Full Text]
74. Jin, H., R. Carrio, A. Yu, T. R. Malek. 2004. Distinct activation signals determine whether IL-21 induces B cell costimulation, growth arrest, or Bim-dependent apoptosis. J. Immunol. 173: 657-665. [Abstract/Free Full Text]
75. Nakajima, H., E. W. Shores, M. Noguchi, W. J. Leonard. 1997. The common cytokine receptor chain plays an essential role in regulating lymphoid homeostasis. J. Exp. Med. 185: 189-195. [Abstract/Free Full Text]
76. Jameson, S. C.. 2002. Maintaining the norm: T-cell homeostasis. Nat. Rev. Immunol. 2: 547-556. [Medline]
77. Boise, L. H., A. J. Minn, C. H. June, T. Lindsten, C. B. Thompson. 1995. Growth factors can enhance lymphocyte survival without committing the cell to undergo cell division. Proc. Natl. Acad. Sci. USA 92: 5491-5495. [Abstract/Free Full Text]
78. Berard, M., K. Brandt, S. Bulfone-Paus, D. F. Tough. 2003. IL-15 promotes the survival of naive and memory phenotype CD8⁺ T cells. J. Immunol. 170: 5018-5026. [Abstract/Free Full Text]
79. Melchionda, F., T. J. Fry, M. J. Milliron, M. A. McKirdy, Y. Tagaya, C. L. Mackall. 2005. Adjuvant IL-7 or IL-15 overcomes immunodominance and improves survival of the CD8⁺ memory cell pool. J. Clin. Invest. 115: 1177-1187. [Medline]
80. Opferman, J. T., B. T. Ober, P. G. Ashton-Rickardt. 1999. Linear differentiation of cytotoxic effectors into memory T lymphocytes. Science 283: 1745-1748. [Abstract/Free Full Text]
81. Gett, A. V., F. Sallusto, A. Lanzavecchia, J. Geginat. 2003. T cell fitness determined by signal strength. Nat. Immunol. 4: 355-360. [Medline]
82. Hou, S., L. Hyland, K. W. Ryan, A. Portner, P. C. Doherty. 1994. Virus-specific CD8⁺ T-cell memory determined by clonal burst size. Nature 369: 652-654. [Medline]
83. Iezzi, G., K. Karjalainen, A. Lanzavecchia. 1998. The duration of antigenic stimulation determines the fate of naive and effector T cells. Immunity 8: 89-95. [Medline]
84. Sivakumar, P. V., D. C. Foster, C. H. Clegg. 2004. Interleukin-21 is a T-helper cytokine that regulates humoral immunity and cell-mediated anti-tumor responses. Immunology 112: 177-182. [Medline]
85. Holm, C., C. G. Nyvold, S. R. Plaudan, A. R. Thomsen, M. Hokland. 2006. Interleukin-21 mRNA expression during virus infections. Cytokine 33: 41-45. [Medline]
86. Brandt, K., S. Bulfone-Paus, A. Jenckel, D. C. Foster, R. Paus, R. Ruckert. 2003. Interleukin-21 inhibits dendritic cell-mediated T cell activation and induction of contact hypersensitivity in vivo. J. Invest. Dermatol. 121: 1379-1382. [Medline]
87. Ma, H. L., M. J. Whitters, R. F. Konz, M. Senices, D. A. Young, M. J. Grusby, M. Collins, K. Dunussi-Joannopoulos. 2003. IL-21 activates both innate and adaptive immunity to generate potent antitumor responses that require perforin but are independent of IFN-. J. Immunol. 171: 608-615. [Abstract/Free Full Text]
88. Ugai, S., O. Shimozato, K. Kawamura, Y. Q. Wang, T. Yamaguchi, H. Saisho, S. Sakiyama, M. Tagawa. 2003. Expression of the interleukin-21 gene in murine colon carcinoma cells generates systemic immunity in the inoculated hosts. Cancer Gene Ther. 10: 187-192. [Medline]
89. Ugai, S., O. Shimozato, L. Yu, Y. Q. Wang, K. Kawamura, H. Yamamoto, T. Yamaguchi, H. Saisho, S. Sakiyama, M. Tagawa. 2003. Transduction of the IL-21 and IL-23 genes in human pancreatic carcinoma cells produces natural killer cell-dependent and -independent antitumor effects. Cancer Gene Ther. 10: 771-778. [Medline]

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