JOURNAL OF VIROLOGY, Mar. 2007, p. 2187–2195 0022-538X/07/$08.00⫹0 doi:10.1128/JVI.01879-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 81, No. 5
Pseudotyped Single-Cycle Simian Immunodeficiency Viruses Expressing Gamma Interferon Augment T-Cell Priming Responses In Vitro䌤 Yue Peng,1 Fan-ching Lin,1 Paulo H. Verardi,1 Leslie A. Jones,1 Michael B. McChesney,3 and Tilahun D. Yilma1,2* International Laboratory of Molecular Biology for Tropical Disease Agents, School of Veterinary Medicine,1 Department of Medical Microbiology and Immunology, School of Medicine,2 and California National Primate Research Center and Department of Pathology, School of Medicine,3 University of California, Davis, California 95616 Received 30 August 2006/Accepted 3 December 2006
To increase the safety and efficacy of human immunodeficiency virus vaccines, several groups have conducted studies using the macaque model with single-cycle replicating simian immunodeficiency viruses (SIVs). However, these constructs had poor or diminished efficacy compared to live attenuated vaccines. We previously showed that immunization of macaques with live attenuated SIV with a deletion in the nef gene and expressing gamma interferon (IFN-␥) results in significantly enhanced safety and efficacy. To further enhance safety, we constructed and characterized single-cycle SIVs, pseudotyped with the glycoprotein of vesicular stomatitis virus, expressing different levels of macaque IFN-␥. Expression of IFN-␥ did not alter the infectivity or antigenicity of pseudotyped SIV. The transduction of dendritic cells (DCs) by IFN-␥-expressing particles resulted in the up-regulation of costimulatory and major histocompatibility complex molecules. Furthermore, T cells primed with DCs transduced by SIV particles expressing high levels of IFN-␥ and then stimulated with SIV induced significantly higher numbers of spot-forming cells in an enzyme-linked immunospot assay than did T cells primed with DCs transduced with SIV particles lacking the cytokine. In conclusion, we demonstrated that the transduction of DCs in vitro with pseudotyped single-cycle SIVs expressing IFN-␥ increased DC activation and augmented T-cell priming activity. of small numbers of vaccinated rhesus macaques resulted in a 1- to 3-log reduction of primary viremia after intravenous challenge with pathogenic SIVmac239, but viral loads in the chronic phase of infection in the majority of the animals were indistinguishable from those of control animals (19, 35). As an alternative approach to enhance both the safety and efficacy of live attenuated vaccines, we developed vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped singlecycle SIVs expressing IFN-␥. Pseudotyped HIV-1 generated by the cotransfection of producer cells with one plasmid encoding env-defective HIV-1 proviral DNA and a second plasmid encoding another viral envelope has been shown to go through only one cycle of replication (26, 27), thus enhancing safety yet retaining most of the characteristics of live attenuated vaccines. VSV-G-pseudotyped HIVs have been shown to be 20- to 130-fold more infectious than nonpseudotyped viruses due to CD4-independent entry and broad cell tropism (2). This led to the induction of antibody titers to HIV-1 Gag that were a hundredfold higher as well as increases in T-cell responses to HIV-1 peptides in immunized mice (34). Thus, antigen expression and presentation in target cells are likely to be enhanced. IFN-␥ has intrinsic antiviral activity, up-regulates the expression of major histocompatibility complex (MHC) class I and class II molecules, activates macrophages and NK cells, and has an important regulatory role in directing T-helper 1 (Th1) lymphocyte differentiation (3, 9). Progression to AIDS is often characterized by a loss of Th1 concomitant with increases in Th2 cellular immune responses (14). The adjuvant effects of IFN-␥ on antigen-specific humoral and cellular immune responses have been demonstrated in several animal models (3,
A safe and effective vaccine for human immunodeficiency virus (HIV) is desperately needed to control the pandemic of AIDS. Simian immunodeficiency virus (SIV) infection of rhesus macaques is a model for the development of vaccines and therapeutics for HIV infection and AIDS in humans. A live attenuated virus with a deletion in the nef gene (SIV⌬nef) has been the most effective vaccine in the SIV/macaque model (15, 52). However, its application is restricted since the vaccine virus persists at a low level indefinitely in vaccinated macaques and can be pathogenic to neonatal macaques (5), although pathogenicity in newborn monkeys was shown to be restricted to neonates lacking maternal immunity (52). Additionally, SIV⌬nef can cause disease in adult macaques several years after vaccination (6). Our laboratory constructed and characterized a live attenuated SIV strain (SIVHyIFN) with a deletion in the nef gene and expressing human gamma interferon (IFN-␥) to investigate the potential of the cytokine to enhance the safety and efficacy of live attenuated SIV vaccines. Vaccination of macaques with SIVHyIFN resulted in decreased viral loads and increased resistance to challenge compared to vaccination with SIV⌬nef (23, 25). In an effort to eliminate the risks associated with live attenuated SIV vaccines, several groups constructed single-cycle SIVs as a safer vaccine strategy (18, 19, 35). However, vaccine efficacy was relatively poor (19, 35). Pilot studies
* Corresponding author. Mailing address: International Laboratory of Molecular Biology for Tropical Disease Agents, University of California, Davis, CA 95616. Phone: (530) 752-8306. Fax: (530) 752-1354. E-mail:
[email protected]. 䌤 Published ahead of print on 13 December 2006. 2187
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29, 53). The adjuvant and attenuating activities of other cytokines, such as interleukin-2 (IL-2), are mediated by the induction of IFN-␥ (29, 42). Moreover, IFN-␥ has been used as a therapeutic agent to restore immune function in immunodeficient animals by a retrovirus-mediated gene therapy strategy (51). Therefore, we have chosen IFN-␥ to enhance the immunogenicity of a relatively weak immunogen in the current study. The use of vectors that deliver appropriate cytokines in conjunction with certain antigens is an encouraging approach for inducing potent cell-mediated immunity (CMI) responses and triggering the differentiation of lymphocytes into a Th1 phenotype. On the other hand, excessive production or actions of cytokines can lead to pathologic consequences (22, 30). Thus, we tested different pseudotyped SIVs expressing IFN-␥ at lower or higher levels to elucidate their immunomodulatory effects in an in vitro model. Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) and have the unique ability to prime naı¨ve T cells, playing an important role in the initiation and regulation of immune responses (38, 41). Immature DCs actively capture and process antigens at peripheral sites such as the skin and mucosal surfaces. Upon encountering microbial, proinflammatory, or T-cell-derived stimuli, characteristic phenotypic and functional changes are induced, a process referred to as DC maturation (10). Maturing DCs undergo a rapid burst of cytokine synthesis and expression of costimulatory molecules. These cells then migrate to the T-cell areas of draining secondary lymphoid organs to prime naı¨ve T cells and initiate an adaptive immune response (8). DCs have therefore recently become the target of many vaccine strategies (4, 7) and were utilized as APCs to enhance the stimulation of naı¨ve T cells in vitro in the current study. Our results demonstrate that the expression of IFN-␥ augments DC activation and function. These results are very encouraging for future studies in vivo. MATERIALS AND METHODS Cells, viruses, and media. HeLa, embryonic kidney (293T), A549, and Rat-2 cells were grown at 37°C under 5% CO2 in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and antibiotics (100 U/ml penicillin and 100 g/ml streptomycin), referred to as complete medium. Rhesus macaque peripheral blood mononuclear cells (PBMCs) were maintained in AIM-V medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS. MAGI-CCR5 cells (12) were obtained through the National Institutes of Health AIDS Research and Reference Reagent Program (catalogue no. 3522; obtained from Julie Overbaugh) and were propagated in complete DMEM medium supplemented with 0.25 g/ml amphotericin B, 0.2 mg/ml G418, 0.1 mg/ml hygromycin B, and 1 g/ml puromycin (Sigma, St. Louis, MO). Encephalomyocarditis virus (EMCV) used for the antiviral assay of IFN-␥ was propagated in A549 cells. Aldrithiol-2-inactivated SIVmac239 was provided by Jeffrey D. Lifson, National Cancer Institute AIDS Vaccine Program. Construction of plasmids. pV1EGFP was generously provided by H. Y. Fan (32). This plasmid contains the SIVmac239 genome with deletions in env, vif, nef, and the U3 region, a 700-bp green fluorescent protein (GFP) gene inserted in place of nef, and two premature stop codons in gag (28). In addition, the first two methionine residues of Nef were mutated to threonine to completely block Nef translation. pSIV⌬nef was described previously (25). A KasI-SphI fragment of pV1GFP was replaced with the KasI-SphI fragment of pSIV⌬nef to restore gag-pol and vif, resulting in plasmid pSIV⌬E⌬Ngfp. The macaque IFN-␥ gene was amplified from plasmid pGEM Rac IFN-␥ (50) by standard PCR techniques with Pfu polymerase using primers 5⬘-ATGCTCCGGACGCCACCATGAAAT ATACA-3⬘ and 5⬘-AATTACTCCGGATCACTGGGATGC-3⬘ or 5⬘-ATAACC CGGGCGCCACCATGAAATATACA-3⬘ and 5⬘-AATTAACGGCCGTCACT GGGATGC3⬘ (engineered BspEI, XmaI, and EagI restriction endonuclease
J. VIROL. sites are underlined). The IFN-␥ gene was cloned into the BspEI site of pSIV⌬E⌬Ngfp, generating plasmid pSIV⌬EM␥⌬Ngfp, or into the XmaI-EagI site of pSIV⌬E⌬Ngfp, replacing the GFP gene and generating plasmid pSIV⌬E⌬NM␥. The IFN-␥ gene was also cloned into the BspEI site in the antisense orientation, generating plasmid pSIV⌬EaM␥⌬Ngfp. All nucleotide sequences derived by PCR were confirmed by sequencing using an ABI 3730 capillary electrophoresis genetic analyzer. To produce a mock control supernatant, pLGRN was generated by cloning the GFP gene into the BamHI site of the pLXRN retroviral vector (Clontech, Palo Alto, CA) under the control of the 5⬘ long terminal repeat (LTR) of Moloney murine sarcoma virus. Generation of pseudotyped SIVs. The pseudotyped particles were prepared as follows: 293T cells (90% confluent in 150-cm2 flasks) were cotransfected with one of the pSIV plasmids (35 g) and pVSV-G (18 g; Clontech, CA), which encodes the VSV-G gene under the control of the cytomegalovirus immediateearly promoter using a standard PolyFect transfection protocol (QIAGEN, Valencia, CA) (54). The medium was replaced after 8 to 10 h of incubation. Viral particle-containing media were collected at 48 h, 72 h, and 96 h after transfection, pooled, clarified by centrifugation at 500 ⫻ g for 10 min, and filtered though a 0.45-m-pore-size membrane (Millipore, Billerica, MA). To prepare high-titer stocks, viral particles were concentrated by ultracentrifugation at 72,100 ⫻ g for 90 min (SW 28 rotor). The viral pellets were resuspended in phosphate-buffered saline (PBS) overnight at 4° C. The mock control supernatant was prepared in the same way as described above except that 35 g of pLGRN and 18 g of pVSV-G were used for cotransfection. Viral titration. A transduction assay (54) was performed to determine viral titers. Briefly, HeLa cells were seeded at 5 ⫻ 104 cells per well in 2 ml of complete medium in 6-well plates and incubated overnight. Medium was then removed and replaced by serially diluted viral particles in a total of 0.8 ml of serum-free medium. After 3 h of incubation at 37°C, cells were washed, and 2 ml of complete medium was added. At 48 h after transduction, cells were trypsinized, and the percentage of GFP-positive cells was determined by flow cytometry analysis (FACScan; BD Biosciences, Franklin Lakes, NJ). The titer was calculated as green-forming units (GFU)/ml, according to the formula described previously (54). Viral stocks were also titrated by a MAGI-CCR5 assay as described previously and expressed as transducing units (TU)/ml (12). A third estimate of titer was done using a p27 enzyme-linked immunosorbent assay (ELISA) (Beckman Coulter, Fullerton, CA) according to the manufacturer’s protocol. Immunofluorescence microscopy. To confirm IFN-␥ expression in target cells, HeLa cells were transduced at a multiplicity of infection (MOI) of 1 with different viral particles as described above. Cells were incubated in medium containing monensin sodium (Sigma) at a final concentration of 3 M for the final 5 h of culture. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 at 2 days posttransduction. PBS containing 5% goat serum was used as a blocking solution. Cells were stained using a rabbit anti-rhesus macaque IFN-␥ polyclonal antibody (Abcam Inc., Cambridge, MA) followed by Alexa Fluor 594-conjugated goat anti-rabbit antibody (Molecular Probes, Eugene, OR) diluted in PBS–5% goat serum to stain intracellular IFN-␥. Images were captured using a Nikon Eclipse TS100 microscope. IFN-␥ bioassay. The bioactivity of expressed macaque IFN-␥ was determined by the prevention of cytopathic effects of EMCV on human A549 cells (24). Briefly, 105 HeLa cells in 6-well plates were transduced with different viral particles at an MOI of 0.1 for 2 h. Cells were then washed thoroughly and incubated for 0, 2, 5, or 7 days in a final volume of 2 ml of complete medium. Supernatants were harvested, centrifuged, filtered through 0.2-m filters, and serially diluted in DMEM–5% FBS. Subsequently, 50 l of sample preparations was placed into 96-well plates that were seeded 4 to 6 h previously with 2 ⫻ 104 A549 cells/well in 50 l DMEM–5% FBS. After 24 h of incubation, cells were challenged with the minimal dose of EMCV that gave 100% cytopathic effects, incubated for 24 h, and then stained with crystal violet. IFN-␥ titers (in U/ml) were expressed as the reciprocal of the dilution of sample giving 50% protection against EMCV. DC preparation and flow cytometric analysis. A standard protocol was used to prepare DCs (26, 27). Briefly, PBMCs from healthy donor rhesus macaques were isolated by Ficoll-Hypaque gradient centrifugation, and CD14⫹ cells were positively selected using magnetic beads according to the manufacturer’s protocol (Miltenyi Biotec, Auburn, CA). CD14⫹ cells were cultured in 24-well plates (0.5 ⫻ 106 to 1.0 ⫻ 106 cells/ml) in AIM-V medium supplemented with 1,000 U/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 1,000 U/ml IL-4 (R&D Systems, Minneapolis, MN) for 6 days. The medium was replaced on days 2 and 4. On day 6, cell marker analysis indicated that most of the cells were immature DCs. Immature DCs were then transduced by different viral particles at an MOI of 0.1 or left untransduced in the above-described cytokine medium.
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At 2 h posttransduction, DCs were washed and cultured in the above-described cytokine medium for 2 days to allow the expression of SIV antigens. Samples of supernatants from the DCs were analyzed by ELISA for the presence of SIV p27 (Beckman Coulter) and IFN-␥ (Mabtech, Mariemont, OH) according to the manufacturer’s protocols. For the DC-PBMC coculture experiment, DCs were further stimulated to mature by being cultured in the cytokine medium described above along with tumor necrosis factor alpha (10 g/ml), IL-1 (10 ng/ml), IL-6 (20 ng/ml), and prostaglandin E2 (10⫺6 M) (40) (Sigma). For flow cytometric analysis, loosely attached DCs were harvested by pipetting and stained with phycoerythrin (PE)-conjugated anti-CD80, PE-conjugated anti-CD86, PE-conjugated anti-CD83, allophycocyanin-conjugated anti-CD11c, and allophycocyanin-Cy7-conjugated anti-HLA-DR monoclonal antibodies (BD Pharmingen). Cells were analyzed by using a FACSArray Bioanalyzer (BD Biosciences). A total of 104 events were acquired, and live cells were gated according to forward scattering and side scattering. CD11c was used as a marker to gate monocytederived DCs. The mean fluorescence intensity of each fluorochrome was examined. Data were analyzed using FlowJo software (Tree Star, San Carlos, CA). Coculture of DCs with autologous PBMCs. Nonadherent DCs transduced with different pseudotyped viral particles or mock transduced were harvested 3 days posttransduction, washed, and cocultured with autologous monocyte-depleted PBMCs at a ratio of 1:20 (DCs:PBMCs) in AIM-V medium in the presence of IL-2 (25 U/ml), IL-7 (10 ng/ml), and soluble CD40L (500 ng/ml) (R&D Systems) at 106 cells/ml (16). Half of the medium was replaced with fresh culture medium containing the cytokines mentioned above every 3 days. On day 8, cells were restimulated with a second set of autologous DCs prepared as described above. Seven days later, CD4⫹ or CD8⫹ T cells were positively selected from the cell populations according to the manufacturer’s protocol (Miltenyi Biotec). Fractionated CD4⫹ or CD8⫹ T cells (responder cells) were restimulated with DCs transduced with SIV⌬EaM␥⌬Ngfp/G (abbreviated dSIV) at a ratio of 10:1 (CD4⫹/CD8⫹ T cells:DCs) for 20 to 24 h in an enzyme-linked immunospot (ELISPOT) plate, followed by an IFN-␥ ELISPOT assay. IFN-␥ ELISPOT assay. Ninety-six-well plates (Millipore) were coated overnight at 4° C with 10 g/ml of anti-IFN-␥ monoclonal antibody (Mabtech). The plates were washed and blocked with AIM-V–10% FBS. Mixtures of transduced DCs and responder cells were added to the plates for 20 to 24 h of incubation. Responder cells alone (2.5 ⫻ 104 to 5 ⫻ 104 cells/well), dSIV-transduced DCs alone (2.5 ⫻ 103 to 5 ⫻ 103 cells/well), and untransduced DC-responder cell cocultures were included as negative controls (40). Positive controls were responder cells stimulated with concanavalin A (10 g/ml; Sigma). The plates were then washed with PBS–0.05% Tween 20 and incubated for 1.5 h with 2 g/ml of biotin-conjugated anti-IFN-␥ monoclonal antibody (Mabtech). Wells were washed and incubated for 1 h with 100 l of diluted streptavidin-ALP (Mabtech), followed by BCIP (5-bromo-4-chloro-3-indolylphosphate)-NPT (Roche Biochemicals, Indianapolis, IN) as a substrate. Spot-forming cells (SFC) were counted using a dissecting microscope and normalized to SFC/106 cells. Data analysis. Statistical analyses were performed with the statistical software program GraphPad Prism, version 4.0 (GraphPad Software Inc., San Diego, CA). Data were expressed as the means ⫾ the standard errors of the means (SEM), and a P value of ⬍0.05 was considered significant. The comparative analysis of DCs transduced with different constructs was performed by one-way analysis of variance, followed by Tukey’s multiple comparisons test.
RESULTS Construction of VSV-G-pseudotyped single-cycle SIVs expressing IFN-␥. It is well established that VSV-G can replace the lentivirus Env protein and form pseudotyped virions, expand host cell tropism, and enhance viral infectivity dramatically within a single cycle due to CD4- and chemokine receptor-independent entry and a markedly decreased requirement for Nef (2). This ensures efficient antigen entry and delivery into exogenous and endogenous presentation pathways for Band T-cell activation (34, 39), mimicking infections of cells with live attenuated SIVs without the potential pathogenicity due to virus replication. In contrast to lentiviral vectors developed for gene therapy applications where viral genes were minimally retained (32), our intent was to preserve as many immunogens as possible while ensuring a single round of infection. We thus constructed single-cycle VSV-G-pseudotyped SIVs using a
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FIG. 1. Strategy for generating pseudotyped single-cycle SIVs expressing IFN-␥. pSIV239 encoding the full-length SIVmac239 proviral DNA is shown at the top. Deletions are indicated by shaded regions, and the inserted macaque IFN-␥ and GFP genes are shown as open boxes. IFN-␥ was also inserted in the antisense orientation (designated aM␥). Pseudotyped SIVs were generated by transient cotransfection of pVSV-G and a plasmid encoding the defective proviral DNA into 293T cells.
two-plasmid transient cotransfection system (Fig. 1). As a result, all SIV proteins except Env and Nef are expressed by our constructs. A similar construct (VSV-G-pseudotyped HIV-1) was previously demonstrated to undergo only one round of replication (26). To confirm the single-cycle infectivity of our viral particles, supernatants of primary target cells transduced by virions at an MOI of 1 were used to transduce secondary target cells; no GFP or p27 Gag was detected in secondary target cells after 96 h of incubation (data not shown). Plasmid construction was based on plasmid pV1EGFP (32), since it has the desired segments of the env and nef genes deleted. The Rev-responsive element was preserved to ensure the proper transport of the unspliced and single-spliced RNAs from the nucleus to the cytoplasm. Nef was deleted to eliminate any potential immune-downregulating effects of this protein on the immune response. The macaque IFN-␥ gene was incorporated into the viral genome at the env (pSIV⌬EM␥⌬Ngfp) or nef (pSIV⌬E⌬NM␥) sites (Fig. 1), allowing the expression of the cytokine utilizing the env or nef alternative-splicing signals, respectively. The resulting pseudotyped single-cycle SIVs were designated SIV⌬EM␥⌬Ngfp/G (abbreviated dSIVL␥) and SIV⌬E⌬NM␥/G (abbreviated dSIVH␥). The IFN-␥ sequence was also inserted into the env site in the antisense orientation (pSIV⌬EaM␥⌬Ngfp) to construct a control viral particle (SIV⌬EaM␥⌬Ngfp/G, [abbreviated dSIV]) for the experiments (Table 1). Titers of infectious viral particles were then determined based on GFP expression utilizing the nef gene splicing machinery or Tat transactivation of LTR-lacZ constructs incorporated into MAGI-CCR5 cells (Table 1). As expected, the titers obtained by the two methods were equivalent, since both Nef and Tat are expressed early under the control of the LTR promoter (46). Titers of virions were also determined by a p27 ELISA, which detects infectious as well as noninfectious particles. The relative infectivities for each con-
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J. VIROL. TABLE 1. Titers and infectivities of VSV-G-pseudotyped SIVs
Viral particle
Abbreviation
GFU/mla
TU/mlb
p27 level (ng/ml)c
Infectivity (GFU or TU/ng)d
SIV⌬EaM␥⌬Ngfp/G SIV⌬EM␥⌬Ngfp/G SIV⌬E⌬NM␥/G
dSIV dSIVL␥ dSIVH␥
4.1 ⫻ 106 1.5 ⫻ 106 NAf
NDe 1.6 ⫻ 106 5.0 ⫻ 106
2,417.9 966.7 3,070.0
1,696 1,552/1,655 1,629
a
GFU per milliliter of viral stocks measured on HeLa cells. TU per milliliter of viral stocks measured on MAGI-CCR5 cells. c Nanograms of capsid antigen per milliliter of viral stocks measured by ELISA. d GFU or TU per nanogram of capsid antigen in viral stocks. e ND, not done. f NA, not applicable. b
struct, calculated as GFU or TU per nanogram of p27 in viral stocks (Table 1), were very similar, indicating that IFN-␥ expression did not affect single-round infectivities of the constructs. Biologically active IFN-␥ was expressed at lower or higher levels by different constructs. To confirm IFN-␥ expression in target cells, HeLa cells were transduced with single-cycle SIVs and analyzed by immunofluorescence microscopy at 48 h posttransduction. As shown in Fig. 2A, cells transduced with dSIVH␥ and stained for IFN-␥ by intracellular staining (ICS) had greater fluorescence intensity than those transduced with
FIG. 2. IFN-␥ was expressed at lower and higher levels by different constructs. (A) Untransduced HeLa cells and HeLa cells transduced with dSIV, dSIVL␥, or dSIVH␥ were fixed, permeabilized, and stained with a rabbit anti-rhesus macaque IFN-␥ polyclonal antibody and an Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (red) to demonstrate IFN-␥ expression (lower row). Note that IFN-␥ accumulated in the Golgi apparatus due to monensin treatment. Cells in the upper row were photographed to demonstrate GFP (green) expression. (B) Supernatants of HeLa cells transduced with dSIV, dSIVL␥, or dSIVH␥ at an MOI of 0.1 were collected at different time points. The antiviral activity of macaque IFN-␥ was measured by the prevention of the cytopathic effect of EMCV in human A549 cells. IFN-␥ titers were expressed as the reciprocal of the dilution of sample giving 50% protection against EMCV. The limit of detection is 2 U/ml and is designated by the dotted line. (C) Supernatants of DCs transduced by different viral particles were collected and assayed by IFN-␥ ELISA at 72 h posttransduction. The optical density (O.D.) of each well was read on a standard microplate reader. Results represent samples assayed in duplicate. The background value was subtracted.
dSIVL␥. As expected, dSIV-transduced and untransduced cells showed only background staining. Furthermore, when supernatants of dSIV-transduced HeLa cells were collected and subjected to a standard antiviral assay (Fig. 2B), no IFN-␥ activity was detected. Expression of IFN-␥ by dSIVH␥ and dSIVL␥ was detected by 2 days posttransduction and reached a plateau by 5 days. IFN-␥ gene expression at the nef site (dSIVH␥) resulted in significantly higher (⬃32-fold) antiviral bioactivity than expression of the gene at the env site (dSIVL␥) on day 5. The difference in IFN-␥ expression levels was further confirmed in supernatants of transduced DCs using an ELISA to detect the level of the protein (Fig. 2C). dSIVH␥ transduction resulted in a much higher level of IFN-␥ production in the DC supernatants than dSIVL␥ did at 72 h posttransduction. No IFN-␥ was detected in supernatants of untransduced or dSIVtransduced DCs. These results were in agreement with the different intensities of IFN-␥ ICS, which detected IFN-␥ expression at the single-cell level. The nonreplicating property of the viral particles restricts sustained accumulation of IFN-␥ in the supernatant of transduced cells; this is in contrast to replication-competent SIVHyIFN virus, where higher IFN-␥ bioactivity and persistent accumulation of this protein were demonstrated (25). Equivalent amounts of Gag were produced by single-cycle SIVs in vitro. Our viral titration using GFU or TU was based on SIV early gene expression (Nef and Tat), but viral structural gene products, especially Gag, are the most immunogenic proteins of SIV in vivo. Therefore, we measured p27 Gag concentrations in the supernatants of transduced target cells to compare and delineate viral late gene expression levels as a result of single-cycle infection with different constructs. As indicated in Fig. 3, levels of p27 Gag in the supernatants of transduced HeLa cells, which are responsive to macaque IFN-␥, were not significantly different. No Gag protein was detected in supernatants of untransduced cells. Similar results were found in transduced Rat-2 cells, which are not responsive to macaque IFN-␥. This indicated that the expression of IFN-␥ at early or late stages of the viral replication cycle did not alter Gag expression levels. Similarly, Gag expression was equivalent in transduced DCs, which are nondividing primary cells, in contrast to immortalized cell lines. This is in agreement with our previous results demonstrating that levels of Gag expression by replication-competent SIVs was not affected by the expression of IFN-␥ in cultured cells (25). Antigenicity of pseudotyped SIV was not affected by IFN-␥ expression. To compare the antigenicities of pseudotyped
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FIG. 3. Equivalent amounts of Gag were produced by all singlecycle SIVs in vitro. HeLa, Rat-2, or monocyte-derived dendritic cells were transduced with different constructs at an MOI of 0.1. Two days posttransduction, supernatants were collected, and the p27 Gag concentration was measured by ELISA. The data shown represent the mean values from triplicate samples assayed in duplicate; error bars indicate the SEM.
SIVs, PBMCs from SIV⌬nef-infected rhesus macaques were incubated with equivalent amounts of the different pseudotyped SIVs or aldrithiol-2-inactivated SIVmac239 (10 ng/ml of p27 Gag). In addition, p27 Gag peptide pools, which were previously shown to stimulate SIV-specific memory cell responses (43), were used as a positive control, while PBMCs from naı¨ve macaques were used as negative controls. In each assay, a lack of SIV antigen stimulation (medium alone) was used as a background control. The amount of IFN-␥ produced from transduced cells instead of activated T cells was negligible, as cells from naı¨ve macaques did not have significant numbers of SFC above the background level after stimulation with dSIVL␥ or dSIVH␥ (Fig. 4). Results showed that the antigenicities of the pseudotyped SIVs and aldrithiol-2-inactivated SIVmac239 were not significantly different (Fig. 4). Additionally, the expression of IFN-␥ did not have an effect on antigenicity in this assay. IFN-␥ enhanced antigen presentation by DCs. Previous studies showed that APCs transduced with an IFN-␥ gene responded to this cytokine immunophenotypically and functionally as if the cytokine were exogenously supplied (1, 44). Hence, we hypothesized that pseudotyped single-cycle SIVs expressing IFN-␥ would result in more efficient antigen presentation by the up-regulation of MHC class I/II and costimulatory molecule expression on APCs. To test this hypothesis, we used an in vitro antigen presentation system that has been well characterized (26, 27), where DCs are used as APCs. Peripheral blood was obtained from four naı¨ve macaques, and monocyte-derived DCs were prepared by the cultivation of the monocytes in the presence of GM-CSF and IL-4. The DCs were then transduced with different viral particles at an MOI of 0.1 (titrated on HeLa cells) and evaluated by immunophenotyping. At this MOI, up to 60% of DCs were GFP positive upon transduction, with minimal cell death compared to that at higher MOIs. To exclude the nonspecific modification of DC surface molecules, a mock supernatant control was included. A representative data set from one macaque is shown in Fig. 5A.
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FIG. 4. IFN-␥ expression did not affect the antigenicities of pseudotyped SIVs. PBMCs (2 ⫻ 105 cells/well) from two SIV⌬nefinfected rhesus macaques and five naı¨ve macaques were cocultured with different antigens for 18 h in tissue culture plates and then transferred onto ELISPOT assay plates for another 5 h of incubation. IFN-␥-secreting cells were measured by an ELISPOT assay. Results from two infected macaques and one representative naı¨ve macaque are shown as SFC/106 PBMCs. Columns represent the mean values of cells stimulated by different antigens or medium alone from triplicate experiments assayed in duplicate. Error bars indicate the SEM.
Data from all four macaques demonstrating the increase of the mean fluorescence intensity (MFI) compared to that of the PBS control are shown in Fig. 5B. Expression of CD83, a DC maturation marker; CD86, a costimulatory molecule; and HLA-DR, an MHC class II molecule, on the DC surface was slightly up-regulated in dSIVL␥ and dSIVH␥ groups. Particularly, the expression of CD80, a principal costimulatory molecule for T-cell activation, was significantly higher in the dSIVL␥ (P ⬍ 0.001) and dSIVH␥ (P ⬍ 0.01) groups than in the dSIV group. There was no significant difference in all markers measured between the two IFN-␥ groups. Transduction of DCs with pseudotyped single-cycle SIVs expressing IFN-␥ enhanced T-cell priming responses in vitro. To measure the T-cell priming efficiency of different viralparticle-transduced APCs, immature DCs were prepared by the cultivation of monocytes in the presence of GM-CSF and IL-4. Immature DCs were transduced with different viral particles or a mock control consisting of supernatant from pVSV-G- and pLGRN-cotransfected cells and then induced to differentiate into mature DCs with a previously characterized cytokine cocktail (40) to enable the DCs to fully activate naı¨ve T cells. The mock control was included to delineate T-cell responses generated by VSV-G or other nonspecific antigen stimulations. Autologous naı¨ve PBMCs were mixed with mature DCs at a 20:1 ratio and cocultured. After two rounds of stimulation, CD4⫹ or CD8⫹ T cells purified from the DCPBMC cocultures (responder cells) were restimulated with mature DCs transduced with dSIV at a 10:1 ratio for 24 h, and the IFN-␥-secreting cells were quantified by an IFN-␥ ELISPOT assay. To control for the constitutive expression of IFN-␥ by dSIVL␥- or dSIVH␥-transduced cells as well as any nonspecific activation of responder cells, dSIV-transduced DCs alone, responder cells alone, and responders mixed with untransduced
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FIG. 5. DC activation markers were up-regulated by IFN-␥ expression. Monocyte-derived DCs from four macaques were transduced by various constructs at an MOI of 0.1 or the same volume of PBS for 2 days. DCs were collected and stained with PE-conjugated anti-CD80, anti-CD86, anti-CD83, or allophycocyanin-Cy7-conjugated anti-HLA-DR monoclonal antibodies. Cells were analyzed by flow cytometry, and representative data from one macaque are shown in A. Blue lines indicate histograms of individual surface markers of DCs transduced by different particles; red lines indicate histograms of surface markers of DCs treated with PBS. The relative MFI of each fluorochrome compared to that of PBS-treated cells from four macaques is summarized in B. Columns represent the mean value of the relative MFI of each fluorochrome from four macaque samples. Error bars represent the SEM. * indicates a P value of ⬍0.01 and ** indicates a P value of ⬍0.001 compared to the dSIV group.
DCs at a 10:1 ratio were used as controls. dSIV-transduced DCs alone did not generate significant levels of SFC, while stimulation with concanavalin A (10 g/ml) as a positive control gave rise to too many SFC to count (results not shown). Responder cells alone, primed by IFN-␥-expressing dSIVtransduced DCs, had higher numbers of SFC than did the mock-transduced controls (Fig. 6). This elevation was statistically significant in CD8⫹ responder cells primed by dSIVH␥transduced DCs (P ⬍ 0.05) (Fig. 6B) but not in CD4⫹ responder cells. Due to the single-cycle infectivity of pseudotyped
SIVs and the extensive washing after the transduction of DCs, IFN-␥ produced from dSIVL␥- or dSIVH␥-transduced DCs was unlikely to contribute significantly to the number of SFC in the assay. The efficacy of the antigen priming was shown by the restimulation of responder cells with dSIV (without IFN-␥)-transduced DCs. Both CD4⫹ and CD8⫹ cells primed by dSIVH␥transduced DCs had a significantly enhanced number of SFC compared to cells primed by mock-transduced DCs (P ⬍ 0.05) (Fig. 6A and B) and cells primed by dSIVs (P ⬍ 0.05) (Fig. 6A and B). Although not statistically significant, responder cells
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FIG. 6. Transduction of DCs with IFN-␥-expressing dSIVs enhanced the ability of DCs to prime T cells in vitro. CD4⫹ or CD8⫹ T cells from four naı¨ve rhesus macaques were stimulated twice with mock-, dSIV-, dSIVL␥-, or dSIVH␥-transduced DCs. These cells were then restimulated with dSIV-transduced DCs (black columns) at a ratio of 10:1 for 24 h, and CD4⫹ (A) or CD8⫹ (B) IFN-␥-secreting cells were enumerated by ELISPOT assay. CD4⫹ or CD8⫹ T cells cocultured with untransduced DCs in the same ratio (patterned columns) and CD4⫹ or CD8⫹ T cells alone (blank columns) were included as controls. Data are shown as the mean values of SFC produced by cells from four macaques and assayed in triplicate. Error bars indicate the SEM. Statistical analyses on CD4⫹ or CD8⫹ T-cell responses were performed by parametric analysis of variance, followed by Tukey’s multiple comparisons test. Statistically significant differences (P ⬍ 0.05) between dSIVH␥- and mock-primed cells are indicated by an *; differences between dSIVH␥- and dSIV-primed cells are indicated by **.
primed by dSIVL␥ also had increased numbers of SFC after the final stimulation. This enhancement was antigen specific since responder cells produced less IFN-␥ when stimulated with nonspecific antigen (untransduced DCs). Thus, the transduction of DCs with pseudotyped single-cycle SIVs expressing IFN-␥ augmented their ability to prime T cells, and this correlated with the level of IFN-␥ production by pseudotyped SIVs. DISCUSSION There have been numerous attempts to develop safe and effective vaccines against HIV and AIDS. A recent strategy to enhance both the safety and efficacy of live attenuated AIDS vaccines is the development of single-cycle replication-defective lentiviruses. However, further research must be done before these constructs become highly immunogenic, effective vaccines. Kuate et al. developed nonpseudotyped single-cycle SIVs by a primer complementation strategy and demonstrated that immunized rhesus monkeys developed SIV-specific humoral and cellular immune responses but failed to be pro-
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tected from challenge with pathogenic SIVmac239 (35). Vaccination with these constructs resulted in low levels of anti-Env and anti-p27 Gag antibodies, relatively low CMI responses, and no detectable neutralizing antibodies to Env. This level of immune response may lead to the inadequate suppression of peak and set-point viral loads of challenge viruses and the subsequent loss of control of viral replication. Evans et al. recently constructed a single-cycle SIV strain expressing all viral gene products except Pol using a Gag-Pol complementation strategy (18). Studies with this construct demonstrated that animals with stronger SIV-specific CMI responses generally had lower peak and set-point viral loads and better control of viral replication in the chronic phase of infection (19). To improve both the safety and efficacy of nonreplicating SIV vaccines, we developed VSV-G-pseudotyped single-cycle SIVs expressing IFN-␥. These pseudotyped SIVs were antigenic (Fig. 4), expressed bioactive IFN-␥ (Fig. 2), and were restricted to a single cycle of replication, demonstrating their potential safety and immunogenicity. Additionally, the expression of IFN-␥ did not affect the levels of production of SIV proteins (Fig. 3) and also had a dose-dependent enhancing effect on T-cell priming responses in vitro (Fig. 6). The current proof-of-concept study has focused on the immunoregulating effects of IFN-␥ on CMI responses to Gag. In future studies, the absence of Env can be rectified by boosting with recombinant Env proteins from different strains of SIV. This would generate broadly Env-specific neutralizing antibodies and increase vaccine efficacy. IFN-␥ has attenuating and adjuvant activities when expressed in or combined with recombinant vaccines (20, 24). We and others demonstrated that vaccinia viruses expressing cytokines are attenuated by at least 106-fold in immunodeficient mice (20, 24, 33). A single immunization with vaccinia viruses lacking either the B13R (SPI-2) or the B22R (SPI-1) immunemodulating gene and coexpressing IFN-␥ resulted in undetectable levels of viral replication in vivo without compromising humoral and CMI responses (36). Therefore, we reasoned that IFN-␥ expression by single-cycle SIVs would compensate for the decreased immunogenicity and lead to immune responses equivalent to those induced by live attenuated SIVs. Nevertheless, the use of cytokines as immunomodulators to strengthen immune responses must be well designed and carefully evaluated (13, 22, 30). For example, IL-12, a cytokine that induces the expression of IFN-␥, has been shown to be dose and time dependent when used as an adjuvant (21). To investigate the effects of different doses of IFN-␥, we took advantage of the fact that, in contrast to Nef, which is expressed at an early phase of SIV replication, Env expression is dependent on the accumulation of Tat and Rev (31). Thus, it occurs in the late phase of the cycle (17). We speculated that by using the IFN-␥ gene to replace nef or env, we could express IFN-␥ at different times, which would lead to different levels of IFN-␥ in a single cycle of viral replication. This hypothesis was confirmed by using IFN-␥ ICS, ELISA, and bioactivity assays to show that IFN-␥ was indeed expressed at lower levels when inserted into the env region instead of the nef region (Fig. 2). Additionally, the different timing of expression of IFN-␥ may also play an important role in its effectiveness as an adjuvant or attenuating factor. Therefore, the biological relevance of dif-
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ferent levels of IFN-␥ expression needs to be evaluated in vitro and in vivo in future studies to further delineate this effect. VSV-G pseudotyping of lentiviral vectors presents a convenient approach to prepare high-titer and more stable viruses (11), increasing the potential to achieve high levels of viral gene expression after a single round of infection. Furthermore, it expands host cell tropism to virtually all cells encountered, increasing the efficacy of antigen presentation by not targeting CD4⫹ cells only. In contrast to natural virus infection, the majority of viral gene products are now expressed in cells typically not infected by SIV, thus avoiding the detrimental effects of viral proteins on CD4⫹ T cells that may occur with live attenuated SIVs or other nonpseudotyped single-cycle SIVs. Moreover, VSV-G pseudotyping allows the mucosal administration of vaccine viruses and the potential induction of mucosal immunity due to the natural route of VSV infection and tissue tropism (37). Preexisting immunity to VSV-G in humans/rhesus macaques is quite rare, and the construction of pseudotyped SIVs using different serotypes of VSV-G (New Jersey or Chandipura) in the future could further increase the efficiency of booster immunization by eliminating the interference of high-titer neutralizing antibodies generated against the priming serotype (Indiana) developed here (47). To test the efficiency of IFN-␥-expressing pseudotyped SIVs to prime T-cell responses, DCs were utilized in our in vitro model for their potent antigen-presenting capability. We first evaluated the effect of IFN-␥ expression on DC activation status. CD14⫹ PBMCs were induced to differentiate into immature DCs by GM-CSF and IL-4, followed by transduction with dSIVH␥, dSIVL␥, or dSIV. Measurements of the maturation markers CD80, CD83, CD86, and HLA-DR indicated that, on average, dSIVL␥ induced higher levels of these molecules than did dSIVH␥, although the differences were not statistically significant. Both dSIVL␥ and dSIVH␥ induced significantly higher levels of CD80 than did dSIV (Fig. 5). To investigate the ability of mature DCs activated by pseudotyped SIV transduction to prime T-cell responses, CD14⫹ PBMCs were induced to differentiate into immature DCs. This was followed by transduction with the three pseudotyped SIVs or a mock transduction control in conjunction with additional proinflammatory mediators to elicit further maturation of the DCs. These DCs were then used to prime naı¨ve T cells. After two rounds of stimulation, responder cells cocultured with dSIVL␥- or dSIVH␥-transduced DCs secreted higher levels of IFN-␥ than did responder cells stimulated with dSIV or mocktransduced DCs. This was highest in the CD8⫹ responder cells. Since our constructs do not produce infectious particles, we theorized that augmented IFN-␥ expression was due primarily to enhanced CD8⫹ T-cell activation by antigen priming. This hypothesis was confirmed by a final stimulation of responder cells with dSIV (without IFN-␥)-transduced DCs. This resulted in increased levels of IFN-␥ secretion from responder cells primed by dSIVL␥- and dSIVH␥-transduced DCs, although this was significant only in cells primed by dSIVH␥-transduced DCs. Since this enhancement appeared to be IFN-␥ dose dependent, this suggests that the higher levels of IFN-␥ enhanced the ability of the DCs to activate T cells, although there was no significant difference in the immunophenotypes of DCs transduced with dSIVL␥ or dSIVH␥ (Fig. 5). This may be due to IFN-␥ acting synergistically in a dose-dependent manner with
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proinflammatory cytokines or other T-cell-derived cosignals, such as CD40-ligand, during the interaction of DCs and T cells. This interaction could further enhance DC maturation, antigen presentation by DCs, and Th1 cell differentiation (16, 44, 48, 49). Overall, the CD8⫹ T cells had increased activation compared to the CD4⫹ T cells, consistent with the fact that endogenously expressed viral antigens are more efficiently processed and presented through the MHC class I pathway. Langerhans cells (immature DCs) occupy a significant percentage of the surface area in the epidermis and mucosal surfaces (45) and are a critical component of immune surveillance. Results from this in vitro model are likely to have significant biological relevance since natural host cells targeted by VSV-G are located mainly in the stratum spinosum, where Langerhans cells reside, increasing the chances of transducing these cells. Additionally, the route of immunization can increase the chances of antigen presentation by DCs in vivo, and these cells can also be specifically targeted to enhance immune responses. In conclusion, this study indicates that IFN-␥ expressed in a single-cycle SIV can function as an adjuvant to enhance T-cell priming responses and DC maturation in vitro. The lack of potential progeny virus-associated pathogenesis, significantly enhanced single-round viral infectivity, and antigen presentation mediated by VSV-G as well as the adjuvant effects of IFN-␥ all combine to make this a potentially safer and more efficacious vaccine strategy for AIDS. The use of DCs as APCs provides an opportunity to conduct preliminary studies of T-cell responses to viral antigens in vitro. These results indicate the potential efficacy of these immunogens and aid in designing future experiments in nonhuman primates. Moreover, it will provide important data for developing future lentiviral vectors expressing immunomodulatory genes for vaccines or immunotherapies. ACKNOWLEDGMENTS We thank the members of the International Laboratory of Molecular Biology for Tropical Disease Agents, especially Julia Collins, Kenneth Chan, Shirley Leung, Lael Brown, and Colleen Tang, for their assistance; Carol Oxford for assistance with the flow cytometric study; Hung Y. Fan from University of California—Irvine for providing plasmid pV1EGFP; and the Immunology Core Laboratory of the California National Primate Research Center for assistance with the CMI study. This work was supported by National Institutes of Health grants AI47025, AI53811, AI54951, and AI66344 to T.D.Y. and AI59185 to P.H.V. Y.P. received support from the Jastro Shields Scholarship and the University of California—Davis Humanities Graduate Research Award. REFERENCES 1. Ahuja, S. S., M. R. Brown, T. A. Fleisher, S. K. Ahuja, and H. L. Malech. 1996. Autocrine activation of hemopoietic progenitor-derived myelo-monocytic cells by IFN-gamma gene transfer. J. Immunol. 156:4345–4353. 2. Aiken, C. 1997. Pseudotyping human immunodeficiency virus type 1 (HIV-1) by the glycoprotein of vesicular stomatitis virus targets HIV-1 entry to an endocytic pathway and suppresses both the requirement for Nef and the sensitivity to cyclosporin A. J. Virol. 71:5871–5877. 3. Anderson, K. P., E. H. Fennie, and T. Yilma. 1988. Enhancement of a secondary antibody response to vesicular stomatitis virus “G” protein by IFN-gamma treatment at primary immunization. J. Immunol. 140:3599–3604. 4. Andrews, D. M., C. E. Andoniou, A. A. Scalzo, S. L. van Dommelen, M. E. Wallace, M. J. Smyth, and M. A. Degli-Esposti. 2005. Cross-talk between dendritic cells and natural killer cells in viral infection. Mol. Immunol. 42:547–555. 5. Baba, T. W., Y. S. Jeong, D. Pennick, R. Bronson, M. F. Greene, and R. M. Ruprecht. 1995. Pathogenicity of live, attenuated SIV after mucosal infection of neonatal macaques. Science 267:1820–1825. 6. Baba, T. W., V. Liska, A. H. Khimani, N. B. Ray, P. J. Dailey, D. Penninck,
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