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CD40L-Containing Virus-Like Particle as a Candidate HIV-1 Vaccine Targeting Dendritic Cells

Franco, David PhD*; Liu, Weimin MD†; Gardiner, David F MD*; Hahn, Beatrice H MD†‡; Ho, David D MD*

JAIDS Journal of Acquired Immune Deficiency Syndromes: 15 April 2011 - Volume 56 - Issue 5 - pp 393-400
doi: 10.1097/QAI.0b013e31820b844e
Basic and Translational Science

The central role of dendritic cell (DC) in mounting an immune response to a novel antigen is now well established. We sought to demonstrate the use of a particular vaccine strategy based on directing HIV-1 Gag proteins to DCs in conjunction with an activation signal. CD40L was expressed on the surface of virus-like particles (VLPs) to target HIV-1 Gag antigens to the CD40 receptor on DCs, whereas CD40L-CD40 interaction would also result in cellular activation. Multiple CD40L VLP constructs were made and evaluated in vitro and in vivo. Indeed, one VLP that expressed CD40L to the highest level showed greatest capacity to activate DCs in vitro. Correspondingly, this CD40L-VLP also proved to be most immunogenic in mice in raising both humoral and cellular responses to HIV-1 Gag. Confirmatory studies were performed to demonstrate the increased immunogenicity of CD40L-VLP is no longer observed when tested in CD40-/- mice. Our findings lend support to the belief that vaccine strategies that both target and activate DCs could yield a superior immune response.

From the *Aaron Diamond AIDS Research Center, The Rockefeller University, New York, NY; and Departments of †Medicine and ‡Microbiology, University of Alabama at Birmingham, Birmingham, AL.

Received for publication October 29, 2010; accepted December 8, 2010.

This study was supported by a grant from the Bill & Melinda Gates Foundation as a part of the Collaboration on AIDS Vaccine Discovery grants 38648 and 38643.

The authors have no conflicts of interest to disclose.

Correspondence to: David Franco, PhD, Aaron Diamond AIDS Research Center, The Rockfeller University, 455 First Avenue, New York, NY 10016. (e-mail:

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's web site (

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There is an urgent need to develop a safe, effective, and affordable vaccine against HIV-1 for worldwide use.1 The development of such a vaccine has proven to be a daunting challenge, largely as a result of HIV-1's refractoriness to antibody neutralization and propensity to mutate.2

Live-attenuated virus vaccines are effective at generating specific cellular and humoral immune responses as well as mediating protection against simian immunodeficiency virus infection.3,4 However, such a live-attenuated virus vaccine for HIV-1 is not feasible because of safety concerns over occasional disease development caused by the attenuated virus or potential for reversion to a pathogenic virus.5 Alternatively, virus-like particles (VLPs), which are self-assembling, nonreplicating, and are similar in size and conformation to intact virions,6-12 may mimic the real virus without the possibility of causing immunodeficiency. VLPs are commonly more immunogenic than subunit or recombinant protein immunogens and are able to stimulate both the humoral and cellular arms of the immune system.13 Furthermore, there has been renewed interest in using HIV-1-like particles as test vaccines, partly because of recent successes using similar strategies for hepatitis B virus and human papilloma viruses.14-20

The orchestration of a coordinated immune response by dendritic cells (DCs) makes them a very attractive target for use in vaccine strategies.21,22 One approach for both targeting and maturing DCs is the use of CD40L. This immunostimulatory surface molecule is primarily expressed on mature CD4+ T cells,23,24 whereas its receptor, CD40, is well expressed on DCs. Interaction between CD40L and CD40 is important for T-cell-dependent B-cell activation and isotype switching.25,26 Binding of CD40L to CD40 modulates the cellular immune response by inducing interleukin-12 production and expression of costimulatory molecules residing on antigen-presenting cells including DCs.23 As a result of the upregulation of costimulatory molecules, antigen-presenting cells are activated, CD4+ T-cell responses are augmented by increased cytokine production,23,26 and CD4-dependent naïve CD8+ T cells are activated in vivo. In addition, genetic fusion of CD40L to a target antigen has been demonstrated to be effective in enhancing the cellular immune responses to the vaccine antigen.27,28 In the present study, we expressed full-length CD40L, or its ectodomain fused to the transmembrane-cytoplasmic tail (TC) region of HIV-1 gp41 or baculovirus surface protein gp64, on HIV-1 VLPs. We first characterized the biologic properties of these VLPs in vitro and then investigated the immune responses to them in mice. To our knowledge, this is the first in-depth immunologic study showing strong humoral and cellular immune responses induced by a CD40L-expressing HIV-1 VLP. Our findings support the notion that DC targeting and activation enhances immune responses to a candidate vaccine.

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Construction of Plasmids

Plasmids were constructed by standard methods; details of the cloning strategies are provided as Supplemental Digital Content 1 ( DNA sequencing was used to verify polymerase chain reaction-amplified segments. Primers are summarized in the supplemental Table (see Supplemental Digital Content 2,

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Virus-Like Particle Production and Purification

After the cloning of the previously described pFastBac Dual DNA constructs, recombinant baculoviruses were generated as directed by the manufacturer. The different VLPs were purified using a previously described method.29,30

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HIV-1 p24 Enzyme-Linked Immunosorbent Assay

The enzyme-linked immunosorbent assay HIV-1 p24 kit, which detects and quantifies the HIV-1 p24 core protein, was used according to the protocol of the manufacturer (Perkin Elmer, Waltham, MA).

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Western Blot

Protein samples (cell lysates) were separated electrophoretically through 10% denaturing sodium dodecylsulfate acrylamide gels and electroblotted onto nitrocellulose membranes. The incorporation of mouse CD40L was determined by incubating the membrane with goat antimouse CD40L monoclonal antibody (R&D Systems, Minneapolis, MN) at 1:1000 dilution followed by detection using horseradish peroxidase-conjugated antigoat antibodies (Sigma-Aldrich, St Louis, MO). Alternatively, samples containing purified VLPs were subjected to a Western blot protocol as described previously.

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Immunofluorescence Studies

Sf9 cells (Invitrogen, Carlsbad, CA) were grown in MatTek 35-mm dishes (MatTek Corporation, Ashland, MA) and fixed with 3% to 4% paraformaldehyde in phosphate-buffered saline (PBS) (pH 7.4) for 20 minutes at room temperature after infection with the different baculovirus-derived VLP constructs. Subsequently, cells were rinsed with PBS, permeabilized with 1% Triton-X in PBS for 5 minutes, and blocked with 1% bovine serum albumin in PBS for 30 minutes. Then the primary antibody (mouse monoclonal antibody against clade B consensus Gag; kindly provided by Y. Huang) and goat monoclonal antibody against mCD40L (R&D Systems, Minneapolis, MN) were added to the plates for 2 hours. After three washes, the secondary antibodies (Alexa 488 antimouse antibody and Alexa 594 antigoat antibody; Molecular Probes, Invitrogen, Carlsbad, CA) were added for 45 minutes at room temperature. The cells were subsequently imaged using a Deltavision microscope.

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Immunoelectron Microscopy Studies

Sf9 insect cells previously transfected with the different VLP constructs (in the pFastBac Dual backbone) were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4. The studies were performed as previously described.31

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Biotinylation of Cell Surface mCD40L Proteins

293T cells (ATCC, Manassas, VA) that were transfected with the different VLP constructs (in pVax backbone) were washed with ice-cold PBS containing 0.1 mM CaCl2 and 1 mM MgCl2, and cell surface proteins were labeled with 0.5 mg/mL EZ-Link Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL) in ice-cold PBS on ice for 30 minutes. The reaction was stopped by washing and incubating the cells in PBS containing 100 mM glycine on ice for 10 minutes. Subsequently, membrane proteins were incubated with goat antimouse CD40L monoclonal antibodies for 30 minutes at room temperature. Streptavidin-horseradish peroxidase was used for the visualization of the CD40L proteins after western blot and sodium dodeclysulfate polyacrylamide gel electrophoresis.

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Generation of Monocyte-Derived Dendritic Cells

We generated monocyte-derived DCs (mDDCs) as previously described.32

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Animals and Immunization Protocols

Female BALB/c (H2d) mice (6-8 weeks old) (Charles River, Kingston, NY) and CD40-/- mice (6-8 weeks old) (kindly provided by R. M. Steinman) were used in the VLP immunization protocol. Mice were immunized two times intraperitoneally with 20 μg of the different VLPs on days 0 and 14.

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Enzyme-Linked Immunosorbent Assay

Direct enzyme-linked immunosorbent assay (ELISA) was used to measure serum HIV-1 p24 antibody titers from immunized mice as previously described.33 A modified ELISA method (sandwich ELISA) was used for comparing CD40L levels on the surface of VLPs as previously described.34,35

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ELISpot Assays for Enumeration of Interferon-γ Spot-Forming Cells

Two weeks after the last immunization, the mice were killed and their spleens were removed. Splenocytes were isolated by standard methods, and single-cell suspensions, depleted of red blood cells, were prepared from freshly isolated splenocytes in culture medium (RPMI 1640 medium supplemented with 10% v/v fetal bovine serum, 100 U/mL penicillin/streptomycin and 1 M HEPES buffer). Interferon (IFN)-γ ELISpot assays were performed using an ELISpot set from BD-Biosciences Pharmingen (San Diego, CA) according to the manufacturer's protocol as previously described.33

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Intracellular Cytokine Staining

Splenocytes from immunized mice were analyzed by intracellular cytokine staining for interleukin 2 (IL-2) and gamma IFN-γ T-cell responses against the peptide pools of HIV-1 p24 as previously described.33

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Tetramer Staining

Two weeks after the last immunization, spleens were harvested aseptically, gently homogenized to form a single-cell suspension, washed, and resuspended to a final concentration of approximately 107 cells/mL. Cells were stained with HIV-1 Gag-specific CD8+ T cell iTAg MHC tetramer PE (Beckman Coulter, Fullerton, CA) followed by multiparametric cytometry analysis to detect the HIV-1 Gag-specific CD8+ T cell population.

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Cytokine Bead Array

mDDCs were isolated as described previously. Twenty micrograms of the different purified VLP constructs were then added to the cells for 24 hours. Subsequently, the concentration of the mouse cytokine IL-12 was measured using the IL-12 cytokine cytokine bead array kit according to the protocol of the manufacturer (BD-Biosciences, PharMingen, San Diego, CA).

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Statistical Analysis

Data were expressed as mean values ± standard deviations using the GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA). An unpaired two-tailed Student t test was performed when comparing two different groups. A P value <0.05 was considered to be statistically significant.

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The Construction, Purification, and Expression of Chimeric HIV-1 Virus-Like Particles Containing Chimeric Mouse CD40L

The incorporation of full-length CD40L in the context of simian immunodeficiency virus VLPs was previously proved to enhance overall immunogenicity.34,35 This finding was recently confirmed using pseudotyped single-cycle simian immunodeficiency virus.36 Based on these observations, we explored the possibility of increasing further the quality and quantity of immune responses against HIV-1 Gag by modifying the immunostimulatory molecule CD40L. One such strategy was to fuse the ectodomain of CD40L37-39 to the TC region of gp41, a molecule that shares the same propensity with CD40L for trimerization.40,41 It was believed that the cytoplasmic tail of gp41 would interact with the HIV-1 Gag p17 matrix protein, which helps to secure the CD40L-gp41 hybrid protein to the surface of the assembling viral particle. A chimeric protein containing the ectodomain of CD40L and the TC region of baculovirus gp64 was also constructed.

As depicted in Figure 1, full-length CD40L or the ectodomain of CD40L (116-250) fused to the TC region of HIV-1 clade B consensus gp41 or baculovirus surface protein gp64 was inserted downstream of the p10 baculovirus late promoter into a pFastBac Dual baculovirus expression vector. HIV-1 clade B consensus Gag was also expressed in the same vector driven by the polyhedrin baculovirus late promoter. Different CD40L variants were also constructed in the pVax and pAdVax backbone vectors (mammalian expression vectors) for CD40L surface expression studies and production of small amounts of chimeric VLPs, respectively. The expression level was subsequently measured with Western blot after transfection of sf9 cells with the plasmid constructs containing the different CD40Ls. The mouse CD40L and the HIV-1 clade B consensus Gag were well expressed by all the different VLP variants studied (Fig. 2A). Subsequently, the surface expression of the CD40L construct was also analyzed using a surface biotinylation assay. A band indicating the presence of biotinylated CD40L molecules was observed after lysis of cells previously transfected with the CD40L/gp41-VLP construct (Fig. 2B: Lane 3). The same result was obtained for other VLP variants (data not shown).

Immunofluorescence studies also confirmed that the CD40L/gp41 VLP construct was well expressed in sf9 cells (Fig. 2C: Panels 4, 5, and 6). Similar results were obtained for the other different VLP constructs (data not shown). Furthermore, CD40L expression was not seen when sf9 cells were transfected with VLPs expressing Gag alone (Fig. 2C: Panels 1, 2, and 3). Immunoelectron microscopy was used to determine the surface expression of full-length CD40L on the chimeric VLPs. In Figure 2E, immunoelectron labeling was observed in the VLPs that expressed CD40L on their surface, as demonstrated by the observed dots indicating the presence of antibodies conjugated with gold particles. No immunoelectron labeling was observed when VLPs expressed only Gag.

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Higher Surface Expression Levels of CD40L on Chimeric Virus-Like Particles Expressing CD40L/gp64

An ELISA assay was used to determine if fusing the TC region of the baculovirus surface protein gp64 to the ectodomain of CD40L would enhance surface expression levels of this molecule. As shown in Figure 2D, the sample containing PBS as well as the one containing VLP without CD40L displayed background levels, whereas all the different chimeric CD40L VLPs expressed CD40L on their surface. The VLP with CD40L/gp64 (CD40L/gp64-VLP) on its surface expressed statistically significant more CD40L molecules than the other chimeric VLPs. This result supports a previous observation that exchanging the TC region from other viral surface proteins such as HIV envelope and influenza hemagglutinin with a similar region of the baculovirus surface protein gp64 increases VLP protein expression.42,43

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Biologic Activity of CD40L Incorporated Into Virus-Like Particles

To determine if the expressed chimeric CD40L molecules on the surface of VLPs are functional, in vitro assays were performed with mDDCs from naive mice. It is well established that the immunostimulatory molecule CD40L is capable of DC maturation.26 The commonly used surface marker indicating the activating status of the DCs is the CD86 molecule. Figure 3A shows the results of such an in vitro dose-dependent activation assay. Incubating mDDCs with different concentrations of the different chimeric CD40L VLPs for 24 hours enhanced the level of CD86 expression. It shows that although expression levels of CD86 were similar for PBS and VLP (approximately 8%-9%), incubating the mDDCs with all the VLPs expressing CD40L on their surface during that same period greatly increased the CD86 expression levels (up to 40%). There was clearly a saturation effect observed when high concentrations of VLP were used. Activation levels after incubating the dendritic cells with lipopolysaccharide were similar to those of all the different chimeric CD40L VLPs (data not shown). Incubating the mDDCs from CD40-/- mice with the different VLP constructs showed that the effect was not only CD40L-dependent, but also CD40-dependent. CD86 levels were comparable to those of PBS and VLP (approximately 12%). Adding 20 μg of the different chimeric VLPs seemed to be enough for full activation of mDDCs; we therefore decided to use this amount of purified VLPs for all the later in vitro and in vivo assays. Although the previous findings clearly showed that the purified VLPs expressing the different CD40L constructs were functional (both CD40- as well as CD40L-dependent), significant differences in mDDC activation status, as measured by CD86 expression level, were not observed.

CD40L is also known to upregulate production of proinflammatory cytokines and chemokines by DCs, especially IL-12, a cytokine responsible for polarizing CD4+ T cells to Th1 type immune responses.44 As shown in Figure 3B, IL-12 expression levels were markedly enhanced after incubating the chimeric CD40L/gp64 VLPs with mDDCs for 48 hours. All other chimeric CD40L VLP constructs failed to do so. This assay, therefore, seems to be more discriminating than the previous described in vitro cell maturation methods, because a clear difference in IL-12 production was recorded when treating the mDCCs with CD40L/gp64 VLPs. This finding suggests that mDDCs only were capable of inducing IL-12 when incubated with VLPs expressing higher expression levels of CD40L (CD40L/gp64 VLP).

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Effect of Virus-Like Particles Displaying CD40L/gp64 on Immune Responses in Mice

To determine if the observed enhancement of CD40L/gp64-VLPs on CD40L expression (Fig. 2D) and cytokine IL-12 induction levels (Fig. 3B) can be translated into an overall improved immune response, BALB/c mice were injected intraperitoneally with 20 μg of the different purified VLPs. Two weeks after priming, a homologous boost was performed. Figure 4A demonstrates that only VLPs expressing the chimeric CD40L/gp64 were able to induce a considerable number of HIV-1 p24-specifc CD8+ IFN-γ-expressing cells. Although the level and quality of antibodies against Gag do not have significance for HIV-1 neutralization, the level of antibody against this protein can still be used to assess relative humoral responses. Figure 4B shows that all the VLPs were able to induce a significant humoral response to p24 with serum titers up to 1/10,000. The CD40L/gp64 VLPs generated more antibodies against p24 than the other VLP constructs, but not by a statistically significant amount.

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Dependence on CD40L and CD40 in CD40L Virus-Like Particle-Mediated Activations

To determine if the described immune response is CD40L- and CD40-dependent, CD40-/- mice kindly provided by R. M. Steinman were used in addition to the wt BALB/c mice. As shown in Figure 4C, only when CD40L/gp64 VLPs were inoculated in BALB/c mice, a considerable HIV-1 p24-specific IFN-γ CD8+ T cell response, with levels comparable to those seen in Figure 4A, was induced. When injecting CD40-/- mice were inoculated with the CD40L/gp64 VLP construct, the earlier observed immune response was absent. This result confirms that this response is mechanism-specific depending on the specific CD40L-CD40 interaction.

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HIV-1 p24-Specific CD8+ and CD4+ T Cell responses After Injecting Mice With the CD40L/gp64 Virus-Like Particle Construct

To determine the level of HIV-1 p24-specific CD8+ and CD4+ T cell response after injecting mice with the different chimeric VLPs, the intracellular cytokine staining method was used. As shown in Figure 5A, CD8+ T cell responses against HIV-1 p24 were mainly detected by IFN-γ expression. The levels of IL-2 and of IL-2 plus IFN-γ expressed by the immune cells when mice were injected with the CD40L/gp64 VLP were comparable to those in mice injected with other VLPs. The HIV-1 p24-specific CD4+ T cell response was greater when mice were exposed to the VLPs expressing CD40L/gp64 on their surface than when exposed to the other VLPs (Fig. 5A). The total levels of IFN-γ, IL-2, and IFN-γ plus IL-2 were higher for the CD40L/gp64 VLP sample, which indicates a qualitatively better immune response at least against Gag. An additional assay, tetramer staining, was used to measure the amount of HIV-1 p24-specific CD8+ T cells. As depicted in Figure 5B, CD40L/gp64 VLP induced a considerably higher amount of HIV-1-specific CD8+ T cells than the other VLPs, confirming the previous results (Figs. 4A and 4C) that the VLPs expressing CD40L/gp64 are able to induce a better immune response against Gag. The abolishment of the previous observed immune responses when immunizing CD40-/- mice with the CD40L/gp64 VLP further confirms previous findings that the immune responses are indeed mechanism-specific depending on the CD40L-CD40 interaction. We can conclude that the remarkably enhanced immune response was probably the result of the increased expression levels of surface CD40L as well as the capacity of this chimeric VLP construct to activate mDDCs to produce cytokine IL-12 in vitro.

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In this study, we have demonstrated that different membrane-bound forms of the immunostimulatory molecule CD40L can be incorporated into VLPs in a functionally active form to enhance immune responses to the HIV-1 clade B consensus Gag antigen. All the chimeric CD40L proteins were incorporated efficiently into HIV-1 VLPs (Fig. 2) after transfecting sf9 insect cells with a plasmid harboring clade B consensus gag as well as the different chimeric mouse CD40L sequences as depicted in Figure 1. Furthermore, we demonstrated that all these CD40L on the particles maintain their biologic activities in vitro (Fig. 3A-B). When investigating the surface expression levels of CD40L, we observed a significant increase of these molecules when expressed in the context of CD40L/gp64-VLPs (Fig. 2D). This is not surprising when we consider previous studies in which a dramatic increase in the expression levels of HIV-1 Env and influenza hemagglutinin proteins was noticed with chimeric constructs using baculovirus gp64.42,43 The maturation of mDDCs, as measured by elevated levels of the activation marker CD86, was quite similar on exposure to the same amount of different VLPs (Fig. 3A). However, increased production of IL-12 by DCs was only detected with exposure to VLPs containing CD40L/gp64 (Fig. 3B). This apparent discrepancy could be partly explained by the sensitivity of the different methods used. In any case, the in vitro activation of DCs by CD40L/gp64 VLP was clearly dependent on both CD40 and CD40L (Fig. 3A-B).

In vivo immunogenicity studies were undertaken to determine if the different chimeric HIV-1 VLPs containing different membrane-bound CD40L molecules were also able to enhance immune responses in BALB/c mice. A homologous prime and boost with 20 μg of CD40L/gp64-VLPs, determined to be the optimal concentration for in vitro maturation studies, was sufficient to induce considerable cellular-mediated immune responses (Fig. 4A). In contrast, the other chimeric VLPs injected into BALB/c mice using the same vaccine regimen were only able to generate an antigen-specific antibody response (Fig. 4B). The better antigen-specific T cell responses induced by CD40L/gp64 VLP is likely explained by the fact that these particles expressed more CD40L on the surface (Fig. 2D) and induced more DC activation in vitro (Fig. 3B). This superior immune response generated against HIV-1 Gag is clearly CD40-dependent, because injecting the mice with the same amount of CD40L/g64 VLPs into CD40-/- mice abrogated the previously observed higher immune responses (Figs. 4C and 5). Furthermore, we have found that CD40L/gp64 VLP was most efficient in stimulating both CD4+ and CD8+ T cell responses as measured by intracellular cytokine production after antigen exposure (Fig. 5A) or by antigen-specific tetramer staining (Fig. 5B).

In summary, we provide evidence in this study that the expression of CD40L on VLPs could indeed target and activate DCs, which in turn results in a better immune response in vivo. Such a DC-targeting/activating strategy should be further explored for the development of vaccines to protect against HIV-1 and other important pathogens.

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We thank H. Rosenblatt for editorial assistance, W. Chen for help with figures, and E. Sphicas (Bio-Imaging Resource Center at The Rockefeller University) for technical support in electron microscopy studies.

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HIV-1; Gag p24; virus-like particle; CD40L; CD40; baculovirus gp64; vaccine; immune response

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