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Journal of Leukocyte Biology logoLink to Journal of Leukocyte Biology
. 2011 Aug;90(2):389–398. doi: 10.1189/jlb.0211068

EBV LMP1, a viral mimic of CD40, activates dendritic cells and functions as a molecular adjuvant when incorporated into an HIV vaccine

Sachin Gupta *, James M Termini *, Liguo Niu *,1, Saravana K Kanagavelu *, Helena Schmidtmayerova *, Victoria Snarsky †, Richard S Kornbluth †,2, Geoffrey W Stone *,3
PMCID: PMC3133438  PMID: 21586676

LMP1, a constitutively active analog of CD40, is shown to function as molecular activator of human dendritic cells and macrophages that can be used as a vaccine adjuvant.

Keywords: macrophages, antigen-presenting cell, attenuated viral vaccines

Abstract

HIV-1 does not significantly activate cellular immunity, which has made it difficult to use attenuated forms of HIV-1 as a vaccine. In contrast, EBV induces robust T cell responses in most infected individuals, perhaps as this virus contains LMP1, a viral mimic of CD40, which is a key activating molecule for DCs and macrophages. Consequently, studies were conducted using LMP1 and LMP1-CD40, a related construct formed by replacing the intracellular signaling domain of LMP1 with that of CD40. Upon electroporation into DCs, LMP1 and LMP1-CD40 mRNAs were sufficient to up-regulate costimulatory molecules and proinflammatory cytokines, indicating that these molecules can function in isolation as adjuvant-like molecules. As a first step toward an improved HIV vaccine, LMP1 and LMP1-CD40 were introduced into a HIV-1 construct to produce virions encoding these proteins. Transduction of DCs and macrophages with these viruses induced morphological changes and up-regulated costimulatory molecules and cytokine production by these cells. HIV-LMP1 enhanced the antigen-presenting function of DCs, as measured in an in vitro immunization assay. Taken together, these data show that LMP1 and LMP1-CD40 are portable gene cassettes with strong adjuvant properties that can be introduced into viruses such as HIV, which by themselves, are insufficient to induce protective cellular immunity.

Introduction

Since Jenner's original description of vaccinia, an attenuated form of the smallpox virus, there has been continued interest in using viruses as vaccines. However, whereas viral vaccines, such as yellow fever 17D, induce strong, protective immunity, viral vaccines for other viruses, such as RSV, do not. This difference has been explained by the presence or absence of virus-encoded immunostimulators: yellow fever 17D strongly activates TLRs [1], whereas RSV fails to do so [2]. These examples suggest that viral vaccines are more likely to be effective if they carry within them their own adjuvant-like immunostimulatory molecules.

In this regard, it is notable that HIV-1 is largely deficient in direct immunostimulatory qualities. HIV-1 infection of cultured myeloid DCs, for example, does not induce DC immunostimulatory functions [3]. Instead, HIV-exposed DCs require exogenous stimuli such as CD40L to become activated [4]. CD40L is normally expressed by activated CD4+ T cells and provides DCs with the strong CD40 stimulation needed to generate effective CD8+ T cell responses [5–7].

Given the importance of including immune-activating molecules in viral vaccines, we were impressed by the strong CD8+ T cell immunity engendered by infection with EBV, a γ-herpesvirus (HHV4). EBV infection is very common and is often manifested clinically as AIM, a generally self-limiting disease. Notably, AIM is characterized by an explosion of CD8+ T cells that recognizes EBV antigens [8]. These anti-EBV CD8+ T cells contribute to the resolution of EBV viremia and persist as memory cells at remarkably high levels for years. For example, in healthy EBV-seropositive subjects, up to 5.5% of circulating CD8+ T cells were identified as EBV-reactive, using tetramers for just a single peptide epitope [9]. Although it has not been fully determined why EBV is so immunostimulatory in vivo, we were intrigued by the functional similarity of the EBV LMP1 to the CD40R. Indeed, LMP1 is viewed as a constitutively activated viral mimic of the CD40R [10]. Consequently, we asked if LMP1 and a related protein, LMP1-CD40, could be used as molecular adjuvants for DCs and macrophages. To a remarkable degree, we found that LMP1 and LMP1-CD40 strongly activated these APCs. This function was maintained when these molecules were inserted into the HIV-1 genome, and the resulting viruses were strongly activating for APCs in vitro. Consequently, LMP1 and LMP1-CD40 serve as portable gene cassettes with adjuvant-like qualities that can be used to improve the cellular immune response to viral vaccines.

MATERIALS AND METHODS

Cells and reagents

Venous blood was obtained from Continental Blood Services (Miami, FL, USA) as anonymous buffy coat donations from HIV-seronegative donors. Initial studies were also performed under an approval from the University of California San Diego Institutional Review Board (San Diego, CA, USA). Informed consent was obtained, consistent with the principles described in the Declaration of Helsinki. PBMCs were isolated by centrifugation over Ficoll-hypaque and subsequently cultured in RPMI 1640 (Thermo Scientific HyClone, Logan, UT, USA), supplemented with 5% human AB serum (Lonza, Allendale, NJ, USA) and 10 mM HEPES (Invitrogen, Carlsbad, CA, USA; complete RPMI 1640). 293T human embryonic kidney cells were cultured in DMEM, supplemented with 10% FBS, 2 mM L-glutamine, and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). TZM-bl cells were obtained through the NIH AIDS Research and Reference Reagent Program (Germantown, MD, USA; Catalog No. 8129, contributed by John C. Kappes and Xiaoyun Wu), cultured in RPMI-1640 medium (supplemented with 10% FBS, 2 mM L-glutamine, and antibiotics), and used to quantify infectious HIV virions as described previously [11].

Plasmid DNAs

Plasmid DNA encoding EGFP was obtained from Dr. Celsa Spina (UCSD, La Jolla, CA, USA). pNL-BaL (termed pHIV in the figure legends) is a macrophage-tropic proviral clone of pNL4-3 containing the CCR5-using envelope from HIV-1BaL and was the kind gift of Roberto Mariani and Ned Landau (New York University School of Medicine, New York, NY, USA). Plasmids were grown in Escherichia coli DH5α and isolated using a plasmid maxi kit (Qiagen, Valencia, CA, USA). To reduce the presence of exogenous-stimulating materials, the plasmids were further cleaned using Triton X-114 extraction as described previously [12].

Construction of LMP1 and LMP1-CD40 adjuvant gene cassettes

cDNA was prepared from Raji B cells (American Type Culture Collection, Manassas, VA, USA), and the LMP1 sequence of EBV was isolated by PCR and found to be identical to the reference sequence (GenBank M58153.1). Related to LMP1 is an artificial fusion protein containing the multimerizing, membrane-associated N-terminus of LMP1 conjoined with the intracytoplasmic domain of CD40, i.e., LMP1-CD40. This membrane-associated intracellular fusion protein mimics the constitutive signaling of CD40 without the need for an external ligand [13–16]. LMP1-CD40 was constructed by fusing DNA encoding the N-terminal 190 aa of LMP1 (beginning with the ATG start codon) with the C-terminal 58 aa of CD40, followed by a TGA translational stop codon. For the preparation of LMP1 and LMP1-CD40 mRNA described below, these sequences were cloned into the pGEM-4Z/A64 vector.

Preparation of HIV-1 virions expressing LMP1 or LMP1-CD40

Levy et al. [17] described the design of replication-competent HIV-1 proviral clones, in which exogenous coding sequences such as EGFP could be inserted just 5′ to nef, followed by an IRES that allows continued translation of the natural Nef protein (see Fig. 1B). Starting with the macrophage-tropic pNL-BaL proviral clone, overlap PCR was used to construct plasmids expressing EGFP (pHIV-EGFP), LMP1 (pHIV-LMP1), or LMP1-CD40 (pHIV-LMP1-CD40). It should be noted that these exogenous genes are expressed in nef-spliced mRNAs, where the nef-spliced mRNAs are very strongly expressed and are the predominant mRNA species in macrophages during the first 24 h following HIV-1 infection [18]. This design maximizes the expression of these exogenous genes, whereas other provirus designs would be expected to result in more modest expression of these transgenes.

Figure 1. Molecular design of immunostimulatory forms of HIV-1.

Figure 1.

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(A) The molecular structure of LMP1 and the LMP1-CD40 chimeric fusion protein. In LMP1, the six-transmembrane N-terminal region (N) enables the formation of LMP1 clusters in the plasma membrane. This clustering is essential for LMP1 activity. In the LMP1-CD40 fusion protein, the cell signaling C-terminal region (C) of LMP1 has been replaced with the signaling domain of the CD40R. (B) The proviral clone designed by Levy et al. [17] to express GFP as part of nef-spliced mRNA, followed by translation of nef using an IRES. To create immunostimulatory forms of HIV-1, LMP1 or LMP1-CD40 was inserted in place of GFP as shown (see text).

Preparation of DCs and macrophages and virus transduction

To isolate monocytes from PBMCs, cells were first adhered to T175 plastic flasks (Corning-Costar, Cambridge, MA, USA) for 24 h in RPMI 1640 containing 10% heat-inactivated human AB serum. Following washing to remove nonadherent cells, the adherent monocytes were harvested by gentle scraping and transferred into a 24-well at a density of 1 × 106 cells/well. To allow differentiation into monocyte-derived macrophages (macrophages), the cultures were incubated for an additional 7 days. To prepare monocyte-derived DCs, the monocytes were cultured in 800 U/ml GM-CSF and 500 U/ml IL-4 (R&D Systems, Minneapolis, MN, USA) for 5 days, adding fresh GM-CSF and IL-4 on Day 3.

Lentiviral transduction of DCs and macrophages and flow cytometry

Infections were initiated by aspirating the supernatant media from the wells containing 106 DCs or macrophages and then adding 100 μl/well media containing 100 ng p24 equivalents of HIV (calculated to be a MOI of 0.1), followed by culture at 37°C for 4 h. Next, the cells were washed twice with RPMI medium and then fed with 2 ml complete RPMI 1640, followed by incubation at 37°C for up to 8 days. The culture supernatants of transduced DCs or macrophages were collected at different time-points and stored at –80°C. For flow cytometry, macrophages were stained on the plates and then harvested by scraping, whereas DCs were first harvested by gentle scraping and then resuspended for staining. Cells were washed in FACS buffer (PBS supplemented with 3% FCS and 0.02% sodium azide) and then stained by fluorochrome-conjugated antibodies (Supplemental Methods). Flow cytometry was performed using a LSRII bioanalyzer (Becton Dickinson, San Diego, CA, USA) and analyzed with the FlowJo software program (Tree Star, San Carlos, CA, USA).

Measurement of DC and macrophage activation by cytokine assays and RT-PCR for chemokine mRNA

For cytokine measurements, supernatants were collected from DC and macrophage cultures 48 h after mRNA electroporation or 7 days after virus infection and stored at –80°C until assay. Concentrations of IL-8, IL-6, IL-1β, TNF, IL-10, and IL-12p70 were measured by CBA (BD Biosciences, San Jose, CA, USA), according to the manufacturer's instructions. Additionally, macrophages were infected for 7 days and then analyzed by RT-PCR to measure steady-state levels of CCL3 (MIP-1α), CCL4 (MIP-1β), and CCL5 (RANTES; Supplemental Methods).

In vitro immunization assay for T cell responses

DCs from a HIV seronegative donor were exposed to different HIV viruses for 6 days, which introduced HIV antigens into these APCs. Autologous T cells were prepared by selection of nonadherent cells from PBMC cultures, resulting in a mixed population of lymphocytes. The DCs were then incubated with these autologous T cells for 12 days in the presence of 5 μM nevirapine to prevent HIV infection of any CD4+ T cells in the lymphocyte population. IL-2 (5 U/ml) was added on Day 3 and Day 8. Following DC-T cell coculture, antigen-specific T cell responses were quantified by IFN-γ ELIPOT assay [19]. Cultured cells (105/well) were added to 96-well multiscreen plates (Millipore, Bedford, MA, USA) that had been precoated with 0.5 g/ml anti-IFN-γ mAb (BD Biosciences). A pool of 15-mer Gag HIV-1 Clade B consensus peptides (NIH AIDS Research and Reference Reagent Program, Catalog No. 8117) was added at a final concentration of 5 μg/ml. As a negative control, cells were cultured without peptide. Plates were incubated overnight at 37°C, 5% CO2, and developed as described previously [12]. The numbers of spots were determined using an automated ELISPOT plate reader (CTL Technologies, Cleveland, OH, USA), and the SFC were calculated by subtracting the negative control wells (mean+3 sd). A value of 55 SFC/106 PBMC or greater after subtraction of background was considered positive.

Statistics

Data were analyzed using PRISM 4.0 (GraphPad Software, La Jolla, CA, USA) and expressed as the mean ± sem. A P value of <0.05 was considered statistically significant. Experiments were analyzed by one-way ANOVA, followed by Bonferroni post-test analysis to determine statistical differences between groups.

RESULTS

Preparation of LMP1 and LMP1-CD40

The Raji B cell line was used for the preparation of LMP1 cDNA, which encoded a 387-aa protein. To prepare LMP1-CD40, PCR gene construction techniques were used to fuse the nucleotides for the N-terminal 190 aa of LMP1 (GenBank M58153.1) with the C-terminal cytoplasmic tail of human CD40 (Residues 220–277, GenBank NM_001250). The resulting proteins are depicted in Fig. 1A. Notably, LMP1 N-terminal residues form a domain with six transmembrane regions that self-associates in the plane of the membrane, thereby clustering the cytoplasmic tails of these proteins. On the intracellular side of the plasma membrane, the clustered signaling domains recruit adaptor molecules such as TRAFs to initiate downstream signaling events. CD40L is not needed to induce clustering of these receptor mimics, and as a result, LMP1 and LMP1-CD40 are constitutively active [13–16].

Electroporation of mRNAs for LMP1 or LMP1-CD40 activate DCs

LMP1 and LMP1-CD40 are known to be active in B cells [10] and certain epithelial cells [20], but their effects on DCs were not known. Consequently, in vitro-transcribed mRNA for LMP1 or LMP1-CD40 was electroporated into DCs [21] (Supplemental Materials). For comparison, control cultures included DCs electroporated without added mRNA (mock) and DCs electroporated with mRNA for EGFP, an inactive protein. As shown in Fig. 2A, LMP1 or LMP1-CD40 mRNA alone was sufficient to up-regulate CD40 and CD80 and the CD83 maturation marker on DCs, as measured by flow cytometry. CD86 expression, however, was not changed significantly, reflecting its independent regulation in these cells. In parallel with these membrane changes, DCs were also stimulated to secrete cytokines into the media, as measured by CBA (Fig. 2B). LMP1 and LMP1-CD40 induced sizable amounts of TNF, IL-6, and IL-8. A trace amount of IL-1β was produced as well, but essentially, no IL-10 or IL-12p70 was made. The lack of IL-12p70 production is consistent with reports that CD40 stimulation alone is insufficient to cause IL-12p70 production in the absence of a second stimulus, such as bacterial endotoxin/LPS or other TLR agonist [22, 23]. Thus, these data show that LMP1 and LMP1-CD40 are sufficient to activate many important DC functions, even when used alone as unvectored, isolated gene cassettes.

Figure 2. LMP1 or LMP1-CD40 mRNAs alone are sufficient to activate DCs.

Figure 2.

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LMP1, LMP1-CD40, or control GFP mRNAs were introduced into DCs by electroporation. Other controls were DC cultures electroporated without adding mRNA (Mock) and nontransfected, untreated DCs. Following 48 h of culture, cell-surface markers were analyzed by flow cytometry, and cytokine production was analyzed by CBA. *P < 0.05; **P < 0.01; ***P < 0.001, compared with the Mock and GFP mRNA control. (A) LMP1 or LMP1-CD40 RNA up-regulated CD40 and CD83 maturation markers and CD80 costimulatory molecule but not CD86, as measured by MFI. Bars show the means and sem of triplicate wells. (B) LMP1 or LMP1-CD40 RNA up-regulated the secretion of cytokines IL-6, IL-8, and TNF but was unable to up-regulate secretion of IL-1β, IL-10, or IL-12p70. Bars show the means and sem of triplicate wells.

Introduction of LMP1 or LMP-1-CD40 into a HIV-1 proviral construct

Given the DC-activating effects of LMP1 and LMP1-CD40 alone, it was important to determine whether they would retain this function when produced in the context of a viral vector, e.g., HIV-1. Consequently, a proviral clone of pNL4-3, which had been modified to contain the macrophage-trophic envelope gene of HIV-1BaL, yielding a CCR5-using virus that could infect DCs and macrophages, was obtained from Roberto Mariani and Ned Landau. This virus was further engineered using the design of Levy et al. [17], who showed that exogenous coding sequences could be inserted into the HIV-1 genome just 5′ to nef, followed by an IRES that allows continued translation of the Nef protein (Fig. 1B). This results in infection-competent, replicating viruses that express LMP1 (HIV-LMP1) or a LMP1-CD40 fusion (HIV-LMP1-CD40). When the plasmids encoding HIV-LMP1 or HIV-LMP1-CD40 were transfected into 293 cells, the synthesis of correctly sized LMP1 and LMP1-CD40 proteins was demonstrated by Western blotting (Supplemental Fig. 1).

HIV-LMP1 and HIV-LMP1-CD40 stimulate DCs and macrophages to up-regulate immunologically important cell surface molecules

To determine if LMP1 and LMP1-CD40 function when expressed by a viral vector, DCs and macrophages were transduced with HIV-LMP1 or HIV-LMP1-CD40 and cultured for 4 days to allow these viruses to enter the cells and express virally encoded proteins. At this time-point, DCs developed extensive dendritic processes similar to the changes induced by stimulation with cells expressing membrane CD40L [24] (Supplemental Fig. 2). Using flow cytometry analysis, both engineered viruses affected DCs (Fig. 3A) by up-regulating CD40, CD80, and CD83 and had only minor effects on the expression of CD86. This is a similar pattern of DC activation to that produced when LMP1 or LMP1-CD40 mRNAs were electroporated into DCs (Fig. 2A), indicating that the specific functions of these molecules were unchanged by incorporation into a viral vector. In addition, HLA-DR (a MHC-II molecule) and CCR7 were studied. Although HIV-LMP1 and HIV-LMP1-CD40 did not affect HLA-DR expression by DCs and macrophages, both of these engineered viruses up-regulated CCR7 expression. As CCR7 controls the entry of cells into LNs [25], it is plausible that the CCR7 up-regulation produced by HIV-LMP1 or HIV-LMP1-CD40 could allow these transduced cells to migrate to LNs where they could in turn present the viral antigens encoded by the HIV vector.

Figure 3. HIV-LMP1 or HIV-LMP1-CD40 transduction activates DCs and macrophages to express cell maturation- and activation-associated surface molecules.

Figure 3.

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(A) The expression of surface markers on DCs 4 days after transduction. Flow cytometry events were first gated for DCs using forward-scatter and side-scatter. Isotype antibody control staining (gray-filled histograms), HIV-GFP (thin lines), or HIV-LMP1 and HIV-LMP1-CD40 (thick lines) are shown. Transduction with HIV-LMP1 resulted in DC activation and maturation, as measured by increased levels of CD40, CD80, CD86, CD83, and CCR7 expression but not HLA-DR. By comparison, HIV-LMP1-CD40 produced a modest increase in CD40 and CD83 expression and a minimal increase in CD80 and CCR7 expression when compared with HIV-GFP-transduced cells. APC, Allophycocyanin. (B) The expression of surface markers on macrophages 4 days after transduction. Flow cytometry events were first gated for DCs using forward-scatter and side-scatter. Isotype antibody control staining (gray-filled histograms), HIV-GFP (thin lines), or HIV-LMP1 and HIV-LMP1-CD40 (thick lines) are shown. HIV-LMP1 transduction activated macrophages to express higher levels of CD40 and CD83 maturation markers and CD80 and CD86 costimulatory molecules. By comparison, transduction by HIV-LMP1-CD40 was less active and induced increased levels of CD83 and CD86 but not CD40 or CD80. These results are representative of three experiments on three different donors. (C) HIV-LMP1 transduction up-regulated CD40, CD80, CD83, and CCR7 surface markers in DCs, whereas it up-regulated CD40, CD80, CD83, and CD86 expression in macrophages, as measured by MFI. By comparison, HIV-LMP1-CD40 up-regulated CD40, CD83, and CCR7 surface markers in DCs, whereas it up-regulated CD83 and CD86 expression in macrophages. Bars show the means and sem of three independent samples. *P < 0.05; **P < 0.01; ***P < 0.001, compared with the HIV and HIV-GFP control. MDDC, Monocyte-derived DC.

Similarly, macrophages were strongly stimulated by HIV-LMP1 and up-regulated CD40, CD80, CD83, and CD86 expression (Fig. 3B). In contrast, HIV-LMP1-CD40 was not a strong stimulator of macrophages, although CD86 was modestly up-regulated. These changes were mirrored in the morphological changes observed in these cultures. HIV-LMP1-exposed macrophages underwent extensive cell-cell clumping, as reported previously, using CD40L stimulation [26]. In contrast, HIV-LMP1-CD40 induced a more modest degree of clumping (Supplemental Fig. 2). Statistical analysis was also performed on DC and macrophage surface marker MFI (Fig. 3C). HIV-LMP1 and HIV-LMP1-CD40 significantly up-regulated CD40, CCR7, and CD83 MFIs on DCs, and both up-regulated CD83 and CD86 on macrophages. In contrast, only HIV-LMP1 significantly up-regulated CD80 on DCs and, CD40 and CD83 on macrophages. Thus, for these cell-surface proteins, it appears that HIV-LMP1 and HIV-LMP1-CD40 are active in DCs and macrophages, and HIV-LMP1 showed higher levels overall in both cell types.

HIV-LMP1 and HIV-LMP1-CD40 stimulate DCs and macrophages to produce cytokines and chemokines

As further studies of the DC and macrophage cultures shown in Fig. 3, culture supernatants from Day 4 were studied for the secretion of cytokines and chemokines. For DCs (Fig. 4A), HIV-LMP1 significantly increased the production of IL-1β, IL-6, IL-8, IL-12p70, and TNF. In contrast, HIV-LMP1-CD40 was less active than HIV-LMP1 for cytokine induction in DCs. As shown in Fig. 4B, HIV-LMP1 and HIV-LMP1-CD40 induced macrophages to make IL-6, IL-8, and small amounts of IL-12p70, IL-1β, and TNF. In contrast, there were no significant effects on IL-10 production. Serial measurements made on Days 4, 7, and 10 showed that high levels of IL-1β, IL-6, and IL-8 persisted in these cultures, whereas IL-10, IL-12p70, and TNF fell to background levels by Day 10 (data not shown). In addition, steady-state levels of three chemokines [CCL3 (MIP-1α), CCL4 (MIP-1β), and CCL5 (RANTES)] were increased in macrophages infected by HIV-LMP1 and especially HIV-LMP1-CD40, suggesting fine differences in the cell signaling produced by the two viral constructs (Supplemental Fig. 3).

Figure 4. HIV-LMP1 or HIV-LMP1-CD40 transduction activates DCs and macrophages to secrete immunostimulatory cytokines.

Figure 4.

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(A) The effects of viruses on cytokine secretion by DCs. DCs were exposed to viruses at a MOI of 0.1 and cultured for 7 days, and supernatants were collected for analysis by CBA assay. As shown, HIV-LMP1 induced significant increases in IL-6 and IL-8 secretion, modest increases in IL-1β, IL-12p70, and TNF secretion, and no induction of IL-10 secretion. By comparison, HIV-LMP1-CD40 induced a significant increase in IL-6 secretion, reflecting fine differences in the cell signaling induced by the LMP1 versus LMP1-CD40 adjuvant cassettes in DCs. The bars show the means from three independent experiments with DCs from three different donors. *P < 0.05; **P < 0.01; ***P < 0.001, using an unpaired t test compared with the HIV and HIV-GFP control virus. (B) The effects of viruses on cytokine secretion by macrophages. These cells were exposed to viruses at a MOI of 0.1, and supernatants were collected at various time-points for CBA cytokine analysis. For macrophages, HIV-LMP1 induced a significant increase in secretion of IL-1β, IL-6, IL-8, IL-12p70, and TNF but little or no increase in IL-10 production. By comparison, HIV-LMP1-CD40 induced a significant increase in IL-1β, IL-6, IL-8, and TNF secretion. The bars show the means from three independent experiments with macrophages from three different donors.

HIV-LMP1 and HIV-LMP1-CD40 up-regulate the antigen-presenting functions of DCs in an in vitro immunization assay

To provide an initial indicator of the effects of HIV-LMP1 and HIV-LMP1-CD40 on immune responses to the HIV viral vector, an in vitro immunization model was used. This assay relies on the fact that T cells from HIV-uninfected individuals have a low but definite frequency of reactivity to HIV antigens [27]. Consequently, when purified, autologous T cells were cultured on DCs exposed to these viruses and supplemented with IL-2, the rare anti-HIV T cells in the population can be expected to respond to the HIV antigens in the DCs. Using an IFN-γ ELISPOT assay and a pool of HIV Gag peptides, exposure to HIV-LMP1 was found to significantly enhance the in vitro response to Gag antigen (Fig. 5). In contrast, HIV-LMP1-CD40 was no better than control HIV-GFP in this assay. These data predict that HIV-LMP1 will be a strong immunogen in vivo and suggest that it may be more active in this regard than HIV-LMP1-CD40. To evaluate the TH1/TH2 ratio of this response, CBA analysis was performed on the supernatants from the ELISPOT assay. We observed increased IFN-γ levels for HIV-LMP1 (12 pg/ml). In contrast IL-4 and IL-10 cytokine levels for HIV-LMP1 and HIV-LMP1-CD40 were similar to controls and were <2 pg/ml for all samples.

Figure 5. HIV-LMP1 enhances the ability of DCs to present HIV Gag antigen to T cells in an in vitro immunization assay.

Figure 5.

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(A) Schematic of the experimental protocol. DCs from a HIV-seronegative donor were exposed to different HIV viruses for 6 days and washed and then incubated with autologous T cells for 12 days in the presence of nevirapine and IL-2 (5 U/ml), starting on Day 3. Cultures were then restimulated with a consensus Clade B 15-mer Gag peptide pool, and IFN-γ ELISPOT analysis was performed 24 h later. (B) DCs exposed to native HIV infection do not stimulate anti-HIV T cell responses. This is consistent with the known inability of HIV to stimulate strong T cell responses when compared with more effective viral vaccines for other viruses. However, HIV-LMP1 strongly enhanced anti-Gag T cell responses (***P<0.001 by unpaired t test comparing HIV-LMP1 with the HIV and HIV-GFP control virus). This shows the ability of the LMP1 adjuvant gene cassette to convert a poorly immunogenic virus into a strongly immunogenic one. By comparison, HIV-LMP1-CD40 was much less active in this assay, consistent with the overall weaker effect of HIV-LMP1-CD40 in DCs compared with the HIV-LMP1 virus. These results are representative of three experiments on three different donors. SFU, Spot-forming units.

DISCUSSION

These studies were conducted to determine if LMP1 and the fusion protein LMP1-CD40 could be used as immune stimulators for DCs and macrophages. The starting point was the clinical observation that EBV infection elicits extremely strong CD8+ T cell responses directed against its own antigens [8, 9] and immunological studies showing that this virus contains a viral mimic of the CD40R, LMP1, which is constitutively active [10]. LMP1 was known to be active in B cells, but its effects on DCs and macrophages had not been reported previously. The data obtained in this report now show that LMP1 and LMP1-CD40 are activators of these cells and can be inserted into viral vectors, such as HIV, to convert a weakly immunostimulatory virus into a strong immune stimulator that can be used as the basis for a candidate vaccine.

The key finding is that LMP1 and LMP1-CD40 can be used in isolation to stimulate DCs and macrophages. This was accomplished by electroporating mRNAs for these genes into these cells in vitro and observing the pattern of activation that resulted. Costimulatory proteins on the cell surface were up-regulated, and cytokine production was induced. These data confirm prior reports that the transfection of plasmid DNA encoding LMP1 has strong CD40L-like activating effects on B cells [28] and B cell-derived tumor lines [29]. Extending these prior findings, the present report shows for the first time that LMP1 and LMP1-CD40, separate from any vector containing them, can act alone as completely functional immunostimulatory molecules capable of activating DCs and macrophages.

As adjuvant molecules, LMP1 and LMP1-CD40 offer several advantages: first, LMP1 is one of the few immunostimulatory proteins that does not elicit strong CD8+ T cell responses against itself. Although very strong CD8+ T cell responses are directed against the lytic proteins expressed by EBV [30], CD8+ T cell reactivity against LMP1 is difficult to detect [28]. Thus, DCs and macrophages activated by LMP1 (and presumably also LMP1-CD40) are not likely to be rapidly killed by pre-existing or induced CD8+ T cells recognizing peptides from this protein.

Second, LMP1 and LMP1-CD40 have potential advantages over using CD40L as a molecular adjuvant. Prior studies have shown that the inclusion of CD40L into vaccinia [31], canarypox [32], adenovirus [33–35], lentiviral vectors [36], and SIV [37] significantly improved the immunogenicity of these viruses and viral vectors. In all of these cases, the production of virus or VLPs from CD40L-expressing cells leads to virions [38] and VLPs [39, 40] bearing functional CD40L on their membrane surface, which can be problematic. Virions and VLPs bearing surface CD40L can bind indiscriminately to the many cell types that express the CD40R, including B cells and endothelial cells [41]. This binding can have functional consequences and CD40L-bearing viruses, and VLPs have been shown to activate bystander B cells [42, 43] and macrophages [44]. This results in a potential for inducing autoimmunity and other deleterious effects. In contrast, LMP1 and LMP1-CD40 are cell-associated proteins that internally activate the cells that express them; they do not confer a ligand-like ability to activate other cells that come in contact with the cells that express them. Thus, LMP1 and LMP1-CD40L should not disturb the cell-targeting qualities built into a viral vector nor should they lead to widespread and possibly toxic bystander cell activation. As a result, we consider a viral vector bearing these molecules to be “self-adjuvanting”, by which, we mean that the tight linkage in time and space of the vector-expressed antigen to these adjuvant molecules allows these two moieties to serve as a complete immunogen, focusing the immune response on the selected antigen.

In addition to LMP1 and LMP1-CD40, Spencer and co-workers [45] have developed a chemically controlled method of multimerizing the CD40 intracellular signaling domain. In their system, CID, a bivalent chemical, is used to cluster specially modified CD40 domains tethered to the cytoplasmic side of the plasma membrane. Like LMP1 and LMP1-CD40, this approach requires the introduction of gene constructs into cells by plasmid transfection or viral transduction. Unlike LMP1 and LMP1-CD40, the system requires the separate administration of the CID cross-linking molecule so that it attains pharmacologically adequate tissue levels in vivo [45]. This degree of regulatory control in this system is attractive, but it could be achieved in other ways. For example, the LMP1 and LMP1-CD40 system described herein could be regulated using an inducible promoter system developed by Berkhout and co-workers [46, 47]. In their studies, the Tet-On promoter system was optimized to create a HIV construct that replicates only in the presence of doxycycline [46, 47]. Such a conditionally replicating form of HIV-LMP1 or HIV-LMP1-CD40 could be a safer way of using these vaccine candidates. Even safer would be to use a form of HIV or lentivirus that is limited to a single cycle of replication in vivo [48–50]. In theory, almost any viral vector could be modified to include LMP1 or LMP1-CD40 as molecular adjuvants.

The studies reported herein suggest yet other modifications for improved vectored vaccines. CD40 stimulation is highly synergistic with TLR stimulation for the induction of CD8+ T cell responses [51], which raises the possibility of combining LMP1 or LMP1-CD40 with constitutively active forms of TLRs [52, 53]. Also, in vivo studies are needed to determine if the fine differences between LMP1 and LMP1-CD40 signaling [10] will result in meaningful differences in vaccine responses. Finally, it has been shown that the monocyte-derived DCs evaluated in this study are not fully representative of ex vivo myeloid DC function, and there are key differences with plasmacytoid DCs [54]. To address this concern, future studies could include the infection of ex vivo myeloid and plasmacytoid DCs with HIV-LMP1 and HIV-LMP1-CD40 viruses.

In conclusion, this report describes two portable genetic adjuvants, LMP1 and LMP1-CD40, which can be used to activate DCs and macrophages. These molecules have considerable potential for strengthening existing vaccines based on attenuated viruses or viral vectors. In particular, they could be especially important for the design of HIV vaccines, given our finding that these genetic adjuvants can be easily introduced into the genome of this otherwise poorly immunogenic virus.

Supplementary Material

Supplemental Figures and Methods
supp_90_2_389__index.html (930B, html)

ACKNOWLEDGMENTS

G.W.S. is supported by NIH grants 1R21AI078834 and 1K22AI068489 and the University of Miami Medical School. R.S.K. was supported by NIH grants 1R21AI63982 and 1R21AI073240. The initial DNA sequencing and flow cytometry experiments were performed with the support of the Molecular Biology and Flow Cytometry Cores at the University of California San Diego Center for AIDS Research (AI36214), the Veterans Administration, San Diego Health Care System, and the San Diego Veterans Medical Research Foundation. We thank Roberto Mariani and Ned Landau for providing the pNL-BaL proviral clone. The following reagents were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases: TZM-bl from Drs. John C. Kappes and Xiaoyun Wu and Tranzyme, Inc. (Durham, NC, USA), and HIV-1 Con B Gag peptides, complete set.

The online version of this paper, found at www.jleukbio.org, includes supplemental information.

AIM
acute infectious mononucleosis
CD40L
CD40 ligand
CID
chemically induced dimerization
LMP1
latent membrane protein 1
MFI
mean fluorescence intensity
RSV
respiratory syncytial virus
SFC
spot-forming count(s)
VLP
virus-like particle

AUTHORSHIP

G.W.S., S.G., and R.S.K. designed the experiments and analyzed the data; S.G., J.M.T., L.N., S.K.K., and V.S. performed the experiments; H.S. provided critical expertise in macrophage experimental methods; S.G., R.S.K., and G.W.S. wrote the manuscript; and all of the authors approved the manuscript. R.S.K. and G.W.S. are listed as inventors on patent filings related to the use of LMP1 and LMP1-CD40 in vaccines.

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Associated Data

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Supplementary Materials

Supplemental Figures and Methods
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