Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Emory University, Atlanta, Georgia 30329

JOURNAL OF VIROLOGY, Oct. 2003, p. 10850 10861 Vol. 77, No. 20 0022-538X/03/$08.00 0 DOI: 10.1128/JVI.77.20.10850 10861.2003 Copyright 2003, American Society for Microbiology. All Rights Reserved. Enhancement of gp120-specific Immune Responses by Genetic Vaccination with the Human Immunodeficiency Virus Type 1 Envelope Gene Fused to the Gene Coding for Soluble CTLA4 Bishnu P. Nayak, Gangadhara Sailaja, and Abdul M. Jabbar* Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Emory University, Atlanta, Georgia 30329 Received 7 February 2003/Accepted 18 July 2003 DNA vaccines exploit the inherent abilities of professional antigen-presenting cells to prime the immune system and to elicit immunity against diverse pathogens. In this study, we explored the possibility of augmenting human immunodeficiency virus type 1 gp120-specific immune responses by a DNA vaccine coding for a fusion protein, CTLA4:gp120, in mice. In vitro binding studies revealed that secreted CTLA4:gp120 protein induced a mean florescence intensity shift, when incubated with Raji B cells, indicating its binding to B7 proteins on Raji B cells. Importantly, we instituted three different vaccination regimens to test the efficacy of DNA vaccines encoding gp120 and CTLA4:gp120 in the induction of both cellular (CD8 ) and antibody responses. Each of the vaccination regimens incorporated a single intramuscular (i.m.) injection of the DNA vaccines to prime the immune system, followed by two booster injections. The i.m.-i.m.-i.m. regimen induced only modest levels of gp120-specific CD8 T cells, but the antibody response by CTLA4:gp120 DNA was nearly 16-fold higher than that induced by gp120 DNA. In contrast, using the i.m.-subcutaneous (s.c.)-i.m. regimen, it was found that gp120 and CTLA4:gp120 DNAs were capable of inducing significant levels of gp120-specific CD8 T cells (3.5 and 11%), with antibody titers showing a modest twofold increase for CTLA4:gp120 DNA. In the i.m.-gene gun (g.g.)-g.g. regimen, the mice immunized with gp120 and CTLA4:gp120 harbored gp120- specific CD8 T cells at frequencies of 0.9 and 2.9%, with the latter showing an eightfold increase in antibody titers. Thus, covalent antigen modification and the routes of genetic vaccination have the potential to modulate antigen-specific immune responses in mice. DNA vaccines have been shown to be effective in the induction of immune responses in various animal model systems (31, 48, 62, 63). In particular, their role in priming the immune system has proven to be critical for amplifying antiviral immunity in rhesus macaques (2, 3, 6, 43, 48, 61). Despite the successful application of DNA vaccines to induce immunity, efforts to optimize the efficacy of this mode of antigen delivery are critical to realize the full potential of this vaccine technology (58, 63). There are a number of rate-limiting steps in the pathway of immune induction mediated by DNA vaccines, for example, limited transgene expression and lack of easy access to antigen-presenting cells (APC), especially dendritic cells (DCs). Furthermore, the generation of robust antigen-dependent adaptive immunity appears to be largely dependent on the effective induction of innate immunity by vaccines (5, 41, 42, 56, 57, 69). DCs have the remarkable ability to link both innate and adaptive immune systems, thereby amplifying antigen-specific immune responses. Although the precise mechanisms of immune induction by DNA vaccines are not fully understood, it is clear that the antigen-processing pathways (both endogenous and exogenous cross-presentation) of APC (DCs) are * Corresponding author. Mailing address: Department of Microbiology and Immunology, Emory University School of Medicine, Emory Vaccine Center, 954 Gatewood Rd., Atlanta, GA 30329. Phone: (404) 727-8001. Fax: (404) 727-8199. E-mail: jabbar@microbio.emory.edu. Present address: Department of Immunology, The Scripps Research Institute, La Jolla, CA 92137. utilized by DNA-encoded antigens to elicit immune responses (1, 18 21, 28, 31, 37, 52, 55, 67). Thus, the nature, breadth, and magnitude of the immune response are intimately related to the abundance and antigen-presenting capabilities of APC resident in local tissues, which are the targets of DNA vaccination (7, 12, 25, 31, 34, 47, 73). It has been established that the orchestration of effective T-cell immune responses depends not only on antigenic stimuli (T-cell receptor-major histocompatibility complex [MHC]-antigen complexes [signal 1]) but also on a plethora of cell surface proteins (costimulatory molecules [signal 2]) expressed on T cells and APC capable of amplifying T-cell activation (68). Although the components of signal 2 may not be strictly required to induce effective T-cell immunity in models of viral infection (high antigen load) (4, 70), their absence or lack of participation in settings of low antigenic load (DNA vaccine) would lower the threshold for antigen-specific T-cell activation. Among many costimulatory molecules, CD28 has a primary role in the activation of T cells by signaling through the costimulation pathway, which is dependent on its binding to B7 molecules expressed on APC (68). On the other hand, CTLA4, a protein expressed on activated T cells, plays a negative role in dampening the response by binding to the same set of B7 molecules (17, 24). Both CD28 and CTLA4 are type I transmembrane glycoproteins anchored to the plasma membrane performing distinct, but opposing, functions through intracytoplasmic signaling mechanisms (24, 68). Importantly, CTLA4 binds B7 proteins 10850

VOL. 77, 2003 CTLA4 AND HIV-SPECIFIC IMMUNE RESPONSES 10851 more avidly (30, 45), and this property was exploited to design immunomodulatory reagents (e.g., CTLA4-Ig), which served as valuable tools in a number of immunotherapeutic settings (17, 24). In an elegant study, Boyle et al. (11) provided evidence that a DNA vaccine expressing CTLA4:huIgG was capable of inducing robust human immunoglobulin G (huigg)-specific antibody responses in mice. This strategy was also employed to elicit antihemagglutinin antibodies, which provided protection against lethal flu challenge (22). It is intriguing that this approach worked so well in inducing protective antiviral and antitumor immune responses (22, 35), as opposed to immune suppression mediated by CTLA4-Ig (17, 24). It is likely that transgene expression by DNA vaccine in the injected site was not high enough to cause immune suppression but was efficient in the directed delivery of antigen to APC (for example, DCs) for immune induction. Such antigen delivery has the ability to induce the maturation of DCs more efficiently for ultimate participation of the antigen-loaded DCs in the T-cell activation process in the T-cell areas of secondary lymphoid organs. Thus, these studies constitute an elegant demonstration of how antigen modification could circumvent rate-limiting steps inherent in DNA vaccination, thereby mediating antigen-specific immune enhancement. We have been exploring a number of DNA vaccine strategies capable of enhancing antigen-specific immune responses, and these strategies have relied on in vivo expansion of DCs by a hematopoietic growth factor, Flt-3 ligand (66). In this study, we have constructed a chimeric gene encoding the CTLA4: gp120 fusion protein and used this gene as a DNA vaccine to elicit both cellular and humoral immune responses in mice. In vitro experiments demonstrated that CTLA4:gp120 was able to bind to B7 molecules expressed on Raji B cells. Importantly, the vaccination studies revealed that immunization with CTLA4:gp120 DNA induced robust antigen-specific T-and B-cell responses and that this immune enhancement was dependent on the mode of DNA delivery into mice. MATERIALS AND METHODS Construction of DNA vaccines coding for the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins. The plasmids expressing gp120, CTLA4, and CTLA4:gp120 fusion proteins were created by cloning PCR-amplified fragments in frame with the tissue plasminogen activator leader sequence of the eukaryotic expression vector pjw4303 or pngvl-7. The gp120 portion (nucleotides 100 to 1512) of the simian-hiv SHIV 89.6p envelope gene (40) was amplified using the following primers: gp120 5 NheI (5 -TGC GGC GCC GCT AGC TTG TGG GTC ACA GTC TAT TAT GG-3 ) and gp120 3 BamHI (5 -AAG CGG ATC CAT CAC ACT GTT CTT CTC TTT GCC CTC-3 ). The mouse CTLA4 extracellular domain (secreted form) was amplified from plasmid DNA coding for the full-length mouse CTLA4 gene (13, 44) purchased from the American Type Culture Collection (Manassas, Va.) (catalog no. 63221; F41F4) using the following primers: CTLA-4 5 NarI (5 -AGA AGA GGC GCC GAA GCC ATA CAG GTG ACC CAA C-3 ) and CTLA-4 3 BamHI (5 -CCA TAA TAG ACT GTG ACC CAC AAG TCA-3 ). To construct chimeric gene CTLA4:gp120, we performed two rounds of PCR as described previously (54). In the first round, individual gene fragments (extracellular domain of gp120 and CTLA4) were amplified. This was followed by a second round of PCR where equimolar quantities of the gel-purified PCR products were mixed to sew the gene pieces together by using end primers, i.e., both the 5 -end primer of CTLA4 and the 3 -end primer of gp120. The primers were designed so that the fused chimeric genes would carry restriction sites to facilitate directional cloning into DNA vaccine vectors. We used the following primers for the generation of individual fragments of CTLA4 and gp120: (i) CTLA-4 5 NarI (5 -AGA AGA GGC GCC GAA GCC ATA CAG GTG ACC CAA C-3 ); (ii) CTLA-4 3 (J) gp120 (5 -CCA TAA TAG ACT GTG ACC CAC AAG TCA GAA TCC GGG CAT GGT TC-3 ); (iii) CTLA-4 5 (J) gp120 (5 -GAA CCA TGC CCG GAT TCT GAC TTG TGG GTC ACA GTC TAT TAT GG-3 ); and (iv) gp120 3 BamHI (5 -AAG CGG ATC C AT CAC ACT GTT CTT CTC TTT GCC CTC-3 ). The genes encoding gp120 and soluble CTLA4 (sctla4):gp120 were cloned into pjw4303 (59, 60) (a gift from James Mullins via Harriet Robinson) or pngvl-7 (Gene Vector Labs, University of Michigan, Ann Arbor, Mich.). Transfection and protein Western blot. A total of 1.5 10 6 293-T cells were transfected with 10 to 15 g each of the DNA vaccine constructs using the calcium phosphate method (Invitrogen, San Diego, Calif.). Detergent lysates were made from transfected cells, and gp120 and sctla4:gp120 were detected on Western blots using the anti-hiv 89.6P Env antibody (raised in rabbits; a gift of Robert Doms, University of Pennsylvania, Philadelphia) and the horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (heavy and light chains) (secondary antibody) (diluted 1:5,000) (Geno-Tech, St. Louis, Mo.). For the detection of CTLA4 in the fusion protein or CTLA4-Ig (a positive control), we used goat anti-mouse CTLA4 antibody (Invitrogen). Flow cytometric analysis of the HIV envelope glycoproteins binding to Raji B cells expressing B7 proteins. Raji cells were purchased from the American Type Culture Collection and cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) (HyClone, Logan, Utah), 2 mm L-glutamine, 1 mm sodium pyruvate, penicillin (100 U/ml), and streptomycin (100 g/ml) (complete medium). To this end, Raji cells in 50 l of binding buffer (phosphate-buffered saline [PBS] containing 5 mm EDTA and 1% FCS) were mixed with an equal volume of the medium supernatant obtained from each of the transfected cells (293-T) or the fusion protein or CTLA4-Ig and incubated at 4 C for 30 min. We used 2 g/ml of CTLA4-Ig in 50- l reaction mixtures in the assay. In the case of the conditioned media, we have determined the quantities of secreted gp120 proteins and consistently found these proteins to be at 4 g/ml of the medium (66). Thus, 100 to 200 ng of the proteins are used in the assay. After two washes in binding buffer (200 l each time), rabbit anti-gp120 was added (diluted 1:5 in binding buffer) and incubated for 30 min. The cells were washed twice in binding buffer and incubated for 30 min with saturating concentrations of fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG. The cells were washed twice in binding buffer and resuspended in the same buffer (150 l) and fixed with an equal volume of 1% formaldehyde in PBS. The fluorescence intensity was analyzed in a FACScan flow cytometer. We included CTLA4-Ig in the binding assay to assess the expression of B7 proteins on the surfaces of Raji B cells, and this served as a positive control in our experimental design with HIV envelope glycoproteins (e.g., CTLA4:gp120). CTLA4-Ig binding was detected using FITC-conjugated donkey anti-human IgG (Accurate Chemicals, Westbury, N.Y.), instead of anti-gp120 antibody. Mouse anti-human B7-1 monoclonal antibody (MAb) (clone PSRM3.1.1) and anti-b7-2 MAb (clone 2331; BD Biosciences, San Diego, Calif.) were used to determine the expression of B7-1 and B7-2, respectively. We calibrated the FACScan flow cytometer (Becton Dickinson, San Jose, Calif.) with standard beads (Flow Cytometry Standards Corp., San Juan, P.R.) to assess the mean florescence intensities (MFIs) of various proteins in the assay under the same settings. CTLA4-Ig was a kind gift from Christian Larsen (Emory University). Human IgG was purchased from Sigma Chemical Company (St. Louis, Mo.). Animals and immunizations. Female BALB/c mice (6 to 8 weeks old) were obtained from Charles River via the National Cancer Institute Frederick Cancer Facility and maintained in the vivarium at the Yerkes National Primate Research Center (Atlanta, Ga.) according to the guidelines of the Institutional Animal Care and Use Committee. DNA immunizations were performed either by intramuscular (i.m.), subcutaneous (s.c.), or gene gun (g.g.) routes. For i.m. injections, DNA was delivered into each of the anterior tibialis muscles at a concentration of 1 g/ l (100 g of total DNA per mouse) in 50 l of phosphate buffer (ph 7.4) (33). For the s.c. route, a total of 100 g of DNA (in 100 l/mouse) was delivered at the base of the tail using a 30-gauge needle. Vaccination by the g.g. route was performed by the method of Feltquate et al. (25). Peripheral blood mononuclear cells (PBMCs) and serum samples were collected from the control and immunized mice at day 14 and on day 28 for the primary phase as well as at day 12 for the booster phase of the immune response. In all our vaccination experiments, a booster injection was given by various routes on day 35 of the primary immune response phase unless specified otherwise for specific purposes in individual sets of experiments. Preparation of mouse PBMCs and ICC staining assay. Blood samples were collected from the orbital plexus into tubes containing heparin (5 U/ml) in PBS. A layer of Histopaque (1 ml at 37 C) had been placed in the tubes and spun at 2,000 rpm (20 C) for 20 min. The fraction containing PBMCs was collected and washed twice with PBS and once with complete medium (RPMI 1640 containing

10852 NAYAK ET AL. J. VIROL. 10% FCS). We followed the protocol described by Murali-Krishna et al. (49) for analysis of intracellular cytokine (ICC) production in spleen cells. However, our analysis was done in PBMCs to assess the generation and subsequent evolution of gp120-specific CD8 T cells in control and vaccinated mice, providing a longitudinal snapshot of the immune response (39). Briefly, PBMCs (10 5 to 10 6 cells/well) were stimulated in vitro (96-well U-bottom plates) in the presence and absence of gp120 V3 loop peptide (0.1 g/ml) (IGPGRAFYAR) (restricted by H2-D d in BALB/c mice) (8, 72) or an irrelevant peptide (lymphocytic choriomeningitis virus NP118) in 10% RPMI 1640 complete medium with brefeldin A (Golgiplug; Pharmingen), and ICC staining was performed as described previously (66). ELISA. To determine anti-gp120 antibody titers, we used capture enzymelinked immunosorbent assays (ELISAs) as described previously (66). The wells on the Maxisorb plates (Nunc, Naperville, Ill.) were coated with sheep anti-gp120 antibody (50 g/ml) (CLINIQA, Fallbrook, Calif.) in bicarbonate buffer. After a blocking step (5% powdered milk in PBS containing Tween 20), HIV-1 gp140 89.6P expressed from recombinant vaccinia virus (a gift from Robert Doms) was added to the plates for 1 h at 37 C. After three washes, sera collected from the control and immunized mice were serially diluted in PBS containing Tween 20. After a 2-h incubation at 37 C, anti-mouse IgG coupled to biotin (diluted 1:2,000; Sigma) was added to the wells, followed by Neutr Avidin-HRP conjugate (diluted 1:10,000 dilution; Pierce, Rockford, Ill.). The reactions were then developed by adding o-phenylenediamine (0.5 mg/ml) in a solution containing 0.1 M citric acid, 0.2 M NHPO 2, and 0.1% H 2 O 2 to the wells. The reactions were quenched in 50 l of 2 N sulfuric acid, and absorbance was measured at 492 and 650 nm. The reciprocal of the serum dilution showing an optical density reading more than the optical density of vector control sera (0.1) was taken as the ELISA antibody titer. To determine the IgG subclass, we performed the ELISA as described above and used rat anti-mouse IgG1a or rat anti-mouse IgG2a coupled to biotin (Southern Biotechnology Associates, Birmingham, Ala.) as the secondary antibody instead of anti-mouse IgG coupled to biotin. All the procedures were performed according to the manufacturer s instructions. ELISPOT. The ELISPOT assay was performed by the protocol described by Murali-Krishna et al. (49) with some modifications to detect gp120-specific CD8 T cells producing gamma interferon (IFN- ). MultiScreen 96-well sterile filter plates (Millipore) were coated overnight with rat anti-mouse IFN- antibody (clone R4-6A2; Pharmingen, San Diego, Calif.) (4 g/ml) in 100 l of bicarbonate buffer (ph 9.6) at 4 C. The wells were blocked for 2 h at 37 C with 200 l of RPMI 1640 complete medium and washed twice with sterile PBS. Different dilutions of splenocytes in a total volume of 200 l of RPMI 1640 complete medium containing rat anti-mouse CD28 antibody (1 g/ml) were added to each well. The total cell number was kept constant (10 6 ) in each well by adding -irradiated (1,200 rad) syngeneic feeder cells. Cells were stimulated for 36 h either in the presence or absence of a MHC class I-restricted peptide (IGPGRAFYAR) (0.2 g/ml) at 37 C. The plates were then washed five times with washing buffer (PBS containing 0.05% Tween 20) and incubated overnight at 4 C with biotinylated rat anti-mouse IFN- (clone XMG 1.2; Pharmingen) (1 g/ml) in 100 l of dilution buffer (PBS containing 0.05% Tween 20 and 0.05% FCS). The wells were washed five times and then incubated with streptavidin- HRP (Vector Labs, Burlingame, Calif.) (3 g/ml) in 100 l of dilution buffer for 2 h at room temperature. The wells were washed five times with washing buffer, and the spots were developed by adding 100 l of 3-amino-9-ethyl-carbazole (AEC) using the AEC peroxidase substrate kit from Vector Labs. The reaction was terminated by washing the plates under running water, and the spots were counted with a stereo dissecting microscope. Statistical analysis. In all animal experiments, we used five mice per group, and the means and standard deviations were calculated for each group of mice. For antibody titers, we used pooled serum samples from five mice. The significance of different values for the number of gp120-specific CD8 T cells for the different groups was determined by Student s t test. A series of t tests on the different values for groups of mice immunized with CTLA4:gp120 and gp120 DNA were performed to determine whether the observed differences in the outcome demonstrated true differences in the populations or were the result of random sampling error. A P value of 0.05 was considered statistically significant. RESULTS Expression and secretion of gp120 and CTLA4:gp120 fusion protein. The genes encoding gp120 and CTLA4:gp120 (Fig. 1A) were introduced into 293-T cells by transfection to examine their expression. We probed detergent lysates of both cell and medium supernatant fractions of the transfected cells with two different antibodies (anti-env and anti-ctla4) to assess the expression profiles of gp120 and CTLA4:gp120 on Western blots (Fig. 1B and C). As expected, the control vector samples did not react with the anti-env antibody, but this antibody recognized both the intracellular and extracellular forms of gp120 and CTLA4:gp120 (Fig. 1B). CTLA4:gp120 ran slower than gp120 on the gel, and this decrease in mobility is due to the physical linkage of sctla4 to gp120 adding to the increase in mass for CTLA4:gp120. Although both proteins were expressed at similar levels, intracellular gp120 ran rather more heterogenously than the corresponding CTLA4:gp120 or the extracellular forms of gp120 and CTLA4:gp120 (Fig. 1B). This heterogeneity could be due to the maturation status of the protein in the endoplasmic reticulum (ER), where high-mannose oligosaccharides are added to the protein backbone (38, 51). An anti-ctla4 antibody also recognized both intracellular (lysate) and extracellular (medium supernatant) CTLA4: gp120 (Fig. 1C). Importantly, the CTLA4:gp120 fusion protein, despite having CTLA4 at the N terminus as a fusion partner, was secreted as efficiently as native gp120 from mammalian cells. CTLA4:gp120 binds to Raji cells that express B7.1 and B7.2 proteins. It has been shown that CTLA4 binds avidly to B7 proteins (24, 30, 45). A cell-based qualitative in vitro assay was used to determine whether fusion protein CTLA4:gp120 could bind Raji B cells (Fig. 2). For positive controls, CTLA4-Ig and anti-b7 MAb were used to assess the levels of B7 proteins in this cell type. As evidenced by the shifts in the MFI, both CTLA4-Ig and anti-b7 MAb were able to bind Raji B cells and the isotype antibody control did not show any shift in the MFI, indicating specific binding of the reagents, CTLA4-Ig and anti-b7 MAb, to B7 proteins on Raji B cells (Fig. 2). Importantly, the CTLA4:gp120 fusion protein induced an MFI shift, whereas HIV envelope glycoproteins gp120 and gp140, as shown by their inability to shift the MFI, failed to recognize B7 proteins on Raji cells (Fig. 2). Thus, this cell-based assay has revealed that CTLA4 in the CTLA4:gp120 fusion protein was capable of binding to Raji-B cells, which expressed high levels of B7 proteins on the cell surface. Vaccination protocol (dose, route, and time of primary and booster injections) for longitudinal analysis of HIV gp120- specific immune responses in mice. Figure 3 shows a schematic of the DNA vaccination protocol. Having assessed the expression, secretion, and binding properties of gp120 and CTLA4: gp120 in the vitro assays (Fig. 1 and 2), we performed critical in vivo vaccination experiments to assess the efficacy of DNA vaccines encoding gp120 and CTLA4:gp120 in the induction of antigen-specific T-cell (CD8 ) and antibody responses in mice. To this end, we employed three different vaccination regimens and performed longitudinal analyses of immune responses by sampling blood periodically during the primary and booster phases of the immune response. We chose days 14 and 28 to monitor the magnitude of the primary immune response, and booster injections were given on days 35, 65, and 98. The immune response to the booster injection was assessed 12 days after each of the two booster injections. PBMCs were used in a short-term (6-h) peptide stimulation study to determine the frequency of CD8 T cells, and pooled serum samples were used for the determination of antibody titers (66). This longi-

VOL. 77, 2003 CTLA4 AND HIV-SPECIFIC IMMUNE RESPONSES 10853 FIG. 1. Construction of DNA vaccines encoding CTLA4:gp120, gp120, and CTLA4 and their expression in 293-T cells. (A) The DNA vaccine constructs were generated by a PCR protocol described previously (54, 66), and the specificity of the reactions and the primers used to construct the DNA vaccines were discussed in Materials and Methods. Genes in the expression vectors are under the control of the cytomegalovirus (CMV) immediate-early promoter, and an intervening intron (intron A) is present for efficient expression. The tissue plasminogen activator (tpa) signal leader sequence is added to the N termini of gp120 and CTLA4 instead of using their own ER for optimal translocation and secretion of gp120 and CTLA4:gp120. The genes were cloned in pjw4303 or pngvl7, and both vectors direct the synthesis of equivalent amounts of proteins in in vitro assays (data not shown). (B and C) Western blot analysis of the total cell lysate (L) or medium supernatant (S) from cells transfected with the vector alone and the genes encoding gp120 and CTLA4:gp120. The proteins were identified by the HIV-1 envelope-specific antibody (B) or the antibody specific to CTLA4 (C), which reacted to both CTLA4:gp120 and CTLA4-Ig (a positive control). The positions of molecular size markers (in kilodaltons) are indicated to the right of the blots. tudinal analysis has afforded us the opportunity to examine critically the generation and subsequent evolution of gp120- specific immune responses in individual mice. In vaccination regimen I (i.m.-i.m.-i.m.), the i.m. delivery of CTLA4:gp120 DNA elicits modest levels of antigen-specific CD8 T cells with high antibody titers. Figure 4A shows the frequencies of gp120-specific CD8 T cells in mice injected i.m. with DNA vaccines. All vaccinated mice harbored very similar frequencies of gp120-specific CD8 T cells on day 14 of the primary response. On day 12 after the first booster injection, however, there was a twofold increase in the number of gp120-specific CD8 T cells in mice that received gp120 DNA, but CTLA4:gp120 DNA induced a four- to fivefold increase. This modest twofold increase might not account for differences in protein expression levels, as all three vaccinated groups harbored nearly identical numbers of gp120-specific CD8 T cells (0.3%) on day 14 of the primary response. Importantly, a second i.m. booster injection given on day 98 did not elicit a higher CD8 T-cell response (assessed on day 12 after the booster injection) than that seen after the first booster injec-

10854 NAYAK ET AL. J. VIROL. FIG. 2. CTLA4:gp120 binds to Raji cells expressing B7 (B7.1 and B7.2) molecules. The ability of CTLA4:gp120 to bind to B7 molecules was assessed in a cell-based in vitro assay. An aliquot (50 l) of the medium supernatants from transfected cells (gp120, gp140, and CTLA4:gp120) was used in the binding assay. We included CTLA4-Ig to assess the expression of B7 proteins on the surfaces of Raji B cells as a positive control in our experimental design with HIV envelope glycoproteins (e.g., CTLA4:gp120). CTLA4-Ig binding was detected using FITC-conjugated donkey anti-human IgG, instead of anti-gp120 antibody (anti-gp120ab). Mouse anti-human B7-1 MAb (clone PSRM3.1.1) and anti-b7-2 MAb (clone 2331) were used to determine the expression of B7-1 and B7-2, respectively. Fluorescent intensity was analyzed using a FACScan flow cytometer, and fluorescent standard beads were used to calculate linear fluorescent intensity. tion, indicating that a longer rest period did not result in immune enhancement using this vaccination protocol. Also, i.m. delivery of CTLA4 DNA and gp120 DNA from separate plasmids did not improve the immune response to the booster injection, indicating that the physical linkage of both CTLA4 and gp120 as a single fusion protein appears critical to achieve this modest increase in the CD8 response (data not shown). The cumulative data from individual mice from the mouse groups are shown in Fig. 4B. Previous studies have demonstrated that CTLA4 linked to other antigens was capable of inducing robust antibody responses (11, 22). The concept of the present study was based on these elegant experiments (11, 22). Therefore, we examined the gp120 antibody titers in serum samples from the same mice used to assess the CD8 T-cell response (Fig. 4A). Of the three groups of mice, only the mouse group that received the CTLA4:gp120 DNA contained high gp120 antibody titers after the first and second booster injections. However, gp120 DNA induced higher antibody titers after the second booster injection, but the titers remained lower than that obtained in mice immunized with CTLA4:gp120 (Fig. 4C). Taken together, these experiments have demonstrated that covalent modification of antigen has the propensity to modulate or enhance both T- and B-cell immune responses in mice. In vaccination regimen II (i.m.-s.c.-i.m.), two booster injections (the first booster injection given s.c. and the second booster injection given i.m. after the initial i.m. priming) augment both gp120-specific CD8 T-cell and antibody responses. It has been shown that the injection sites of DNA vaccination could play crucial roles in the type and magnitude of the immune response (1, 19, 21, 31, 37, 55, 67). This could be due to the nature and relative abundance of APC at these DNA delivery sites (12, 25, 31, 34, 47, 73). As shown in Fig. 4, DNA vaccines encoding gp120 and CTLA4:gp120 have different effects on the induction of immune responses (cellular [CD8 ] FIG. 3. Protocol for DNA vaccination and analysis of T-cell and antibody responses in mice. DNA vaccines were introduced into mice via different routes (i.m., s.c., and g.g.), and immune responses (CD8 T-cell and antibody responses) were assessed during primary (days 14 and 28) and booster (day 12) phases of immunization. The first booster injection was given on day 35, and the same DNA vaccine construct used for priming the immune system was used (homologous). The second booster injection (gp120 DNA) was given on day 65 for the experiments shown in Fig. 5 and 6. In the i.m.-i.m.-i.m. vaccination regimen (shown in Fig. 4), the mice had longer rest periods between the first and second booster injections (day 98) before PBMCs and sera were analyzed for gp120-specific CD8 T cells and antibodies, respectively. In all our vaccine experiments, we introduced equal amounts of DNA vaccines to eliminate variability in immune responses, and an empty vector was used as a control. PBMCs were stimulated in vitro for 6hinthepresence of gp120 peptide and stained for intracellular IFN- as previously described (66). d14 pri, 14 days after the primary immunization; Post d12, 12 days after the booster injection.

VOL. 77, 2003 CTLA4 AND HIV-SPECIFIC IMMUNE RESPONSES 10855 FIG. 4. CTLA4:gp120 DNA elicits only marginal enhancement in the number of gp120-specific CD8 T cells but induces robust gp120 antibody titers when delivered i.m. The mice were vaccinated with vector alone or with gp120 or CTLA4:gp120 DNA. The values shown were collected 14 days after the primary immunization or 12 days after the first or second booster injection. (A) Frequencies of CD8 T cells making IFN- in control and vaccinated mice during the primary and booster phases of the immune response. The numbers shown in the top right corners of the graphs are the percentages of gp120-specific CD8 T cells in a representative mouse from each of the vaccinated groups. (B) Histogram of the frequencies of gp120-specific CD8 T cells in all mice in the three groups (means standard errors [error bars]). (C) Antibody titers in the pooled serum samples from control and vaccinated mice (gp120 and CTLA4:gp120 DNA). The time points are the same as those used for CD8 T-cell analysis. ELISA was performed to obtain end point titers. The data are representative of two independent experiments with five mice in each group. versus antibody). Therefore, we hypothesized that injection sites could modulate the efficacy of the DNA vaccines on the basis of CTLA4 targeting in mice. To test this notion, we performed the following experiments, and the results are presented in Fig. 5 and 6. Figure 5A shows the frequencies of gp120-specific CD8 T cells in a representative mouse from each of the groups (vector, gp120, and CTLA4:gp120). On day 28 of the primary response, gp120 and CTLA4:gp120 DNA induced increases of 0.35 and 0.5%, respectively, in the number of gp120-specific CD8 T cells. This number was reduced to 0.2% on day 12 after the s.c. booster injection, indicating the absence of an immune response to the booster injection. Instead, it appears that the number of gp120-specific CD8 T cells generated on day 28 after the primary i.m. injection had waned, giving rise to a pool of memory gp120-specific CD8 T cells. As expected, the control group (vector alone) did not elicit any antigen-specific immune response. Surprisingly, on day 12 after the second booster injection (i.m.) (on day 65), the frequency of gp120-specific CD8 T cells reached as high as 10.8% in a mouse immunized with CTLA4:gp120 DNA, and the mouse immunized with gp120 DNA also harbored a relatively high number (3.5%) of specific

10856 NAYAK ET AL. J. VIROL. FIG. 5. Two booster injections (the first given s.c. and the second given i.m.) after i.m. priming (i.m.-s.c.-i.m.) enhance gp120-specific CD8 T-cell and antibody responses. The mice were vaccinated with vector alone or with gp120 or CTLA4:gp120 DNA. The values shown were collected 14 days after the primary immunization or 12 days after the first or second booster injection. (A) Frequencies of gp120-specific CD8 T cells in a representative mouse from each of the control and vaccinated mouse groups at the primary (day 28 [D28]) and booster phases (12 days after the first booster injection [d12 1st boost] and d12 2nd boost) of DNA vaccination. The numbers shown in the top right corners of the graphs are the percentages of gp120-specific CD8 T cells in a representative mouse in each of the vaccinated groups. (B) Histogram of gp120-specific CD8 T-cell frequencies in all mice in the three groups. (C) ELISPOT analysis of gp120-specific CD8 T cells from the spleens (14 days after the second booster injection) of control and vaccinated mice. (D) gp120 antibody titers in pooled serum samples from four individual mice from each group. The data are representative of the results of two independent experiments with five mice in each group.

VOL. 77, 2003 CTLA4 AND HIV-SPECIFIC IMMUNE RESPONSES 10857 CD8 T cells (Fig. 5A and B). In terms of the magnitude of the immune response to the booster injection, the mice immunized with gp120 and CTLA4:gp120 DNA exhibited 17- and 49-fold increases in gp120-specific CD8 T-cell numbers over those generated after the s.c. booster injection (0.2%). This enhanced response is higher than that achieved by the experiments shown in Fig. 4, which showed only modest increases (two- to fourfold) in the immune responses to booster injections. We also performed ELISPOT to examine splenic CD8 T-cell immune responses (Fig. 5C). It is evident that mice immunized with CTLA4:gp120 DNA contained 800 to 900 spot-forming cells (SFCs), whereas the mouse group immunized with gp120 DNA alone had 200 to 300 SFCs. This is in agreement with the analysis of gp120-specific CD8 T cells assessed by the ICC assay in control and vaccinated mice (Fig. 5A and B). The sera collected from all three groups (Fig. 5B) were used to measure gp120 antibody titers at both the primary and booster phases of the immune response (Fig. 5D). There are no antibodies or very few antibodies raised against gp120 on day 28 after the first i.m. injection (primary phase). On day 12 after the first booster injection, anti-gp120 antibodies were barely detectable in sera of the mice immunized with gp120 DNA alone or CTLA4:gp120 DNA. The antibody titer increased significantly 12 days after the second booster dose of gp120 DNA injected i.m. in both groups of mice. Although the mice immunized with gp120 DNA harbored lower antibody titers than the titers of mice immunized with CTLA4:gp120, the differences were not significant (Fig. 5D). Thus, this mode (i.m.-s.c.-i.m.) of DNA immunization induces high CD8 T- cell and antibody responses to HIV-1 gp120. Also, it is intriguing that a booster s.c. injection after the first i.m. injection did not result in the induction of enhanced immune responses (Fig. 5A, B, and D). Taken together, the results of these experiments have revealed that an additional i.m. booster injection after the i.m. priming injection and the first s.c. booster injection is required to significantly alter the breadth and magnitude of both gp120-specific CD8 T-cell and antibody responses in mice. In vaccination regimen III (i.m.-g.g.-g.g.), two booster g.g. immunizations given after the i.m. priming elicit relatively high numbers of gp120-specific CD8 T cells and robust antibody titers. It is clear from the experiments described above that the route of DNA immunization has profound effects on the magnitude of the immune response elicited against HIV-1 gp120. Therefore, we tested the efficacy of g.g. immunization in the induction of gp120-specific immune responses by the DNA vaccines encoding gp120 and CTLA4:gp120. This mode of antigen delivery targets distinct types of APC in the epidermis of the skin (12, 25, 31, 34, 47, 73). As before, we used three groups of mice to test this approach (Fig. 3). Figure 6A and B show the frequencies of CD8 T cells induced in the vaccinated mouse groups. The mice immunized with the vector control, as expected, did not harbor any antigen-specific CD8 T cells. Note that the frequency of gp120-specific CD8 T cells was as high as 2.8% in a representative mouse in the group immunized with CTLA4:gp120 and that this value for mice immunized with gp120 DNA was 0.9%, indicating a threefold increase in gp120-specific CD8 T cells resident in mice vaccinated with CTLA4:gp120 DNA after the second g.g. booster injection. As shown for s.c. booster injection in the vaccination regimen II section above, the first g.g. booster injection did not result in the induction of high numbers of CD8 T cells (Fig. 6A and B). In the group immunized with CTLA4:gp120, induction of the primary immune response itself is efficient and comparable to the level induced after the second g.g. booster injection in the gp120 DNA group. Thus, although the immunity generated was not robust like that generated by the i.m.- s.c.-i.m. DNA vaccination regimen (Fig. 5A and B), two g.g. booster injections following a single primary i.m. injection revealed that gp120 fused to CTLA4 efficiently induced the gp120-specific CD8 T-cell response. Antibody titers were assessed in all three groups of mice (Fig. 6C). Mice that were given the vector or gp120 DNA did not show gp120-specific antibodies in the primary phase of the immune response. Subsequent booster injections did not result in substantial increases in the titers of antibodies that are specific to gp120 in mice immunized with gp120 DNA alone. However, mice that received CTLA4:gp120 DNA exhibited very robust gp120-specific antibody responses even after the first booster immunization. This elevated antibody response was not increased further after the second g.g. booster injection of DNA into mice, and the gp120-specific antibody levels appeared to have reached their maximum levels. The experiments described above revealed that CTLA4: gp120 DNA was capable of inducing high gp120 antibody titers (Fig. 4C, 5D, and 6C). We wanted to further characterize the antibody subclass elicited by each of the vaccination protocols, as they might represent an important parameter in vaccine efficacy studies. Figure 6D shows the analysis of antibody subclasses present in mice vaccinated with CTLA4:gp120 DNA by two immunization routes. Sera from mice on day 12 after the booster injection (Fig. 4C) or from mice given g.g. booster injections (Fig. 6C) were analyzed for the presence of IgG2a (Th1) and IgG1 (Th2) as described in Materials and Methods. In the case of g.g. immunization, there is a two- to threefold increase in the IgG1 titers over that of IgG2a, indicating that the antibody response is skewed predominantly toward Th2, which is consistent with other studies (25). Furthermore, there is a mixed response in mice given a booster injection of CTLA4:gp120 i.m., which is consistent with the studies reported previously (11, 22). Thus, although CTLA4:gp120 DNA induced robust immune responses, the quality of the response (Th1 versus Th2) follows the patterns consistent with the DNA immunization route (i.m. versus g.g.). DISCUSSION In this study, we have provided evidence that HIV-1 gp120 physically linked to the extracellular domain of CTLA4 was able to induce high levels of both gp120-specific CD8 T cells and antibodies compared to the levels induced by native HIV-1 gp120. Our in vitro experiments have demonstrated that the CTLA4:gp120 protein has retained the ability to bind B cells expressing the costimulatory molecules, B7.1 and B7.2. Furthermore, CTLA4:gp120 appears to be assembled in the cell and secreted into the medium as oligomeric complexes (dimers or multimers), whereas gp120 formed monomers or aggregates (66) (data not shown). Recent studies on the crystal structures of both B7 and CTLA4 proteins have revealed that this class of

10858 NAYAK ET AL. J. VIROL. FIG. 6. Two booster g.g. immunizations after i.m. priming (i.m.- g.g.-g.g.) elicit high numbers of gp120-specific CD8 T cells and robust antibody responses. The mice were vaccinated with vector alone or with gp120 or CTLA4:gp120 DNA. The values shown were collected 14 days after the primary immunization or 12 days after the first or second booster injection. (A) Frequencies of gp120-specific CD8 T cells in a representative mouse from each of the control and vaccinated mouse groups. (B) Histogram of gp120-specific CD8 T-cell frequencies in all mice in the three groups. (C) gp120 antibody titers in pooled serum samples from five individual mice from each group. The data are representative of the results of two independent experiments with five mice in each group. (D) Analysis of antibody subclasses (IgG1 and IgG2a) in sera collected from the mice shown in this panel and in Fig. 4C.

VOL. 77, 2003 CTLA4 AND HIV-SPECIFIC IMMUNE RESPONSES 10859 proteins appear to exist predominantly as dimers in solution (36, 53). It is likely that dimeric structures are functionally relevant in the biology of these proteins in the costimulatory signal transduction pathway in vivo (17, 24, 68). The physical and functional properties of CTLA4:gp120 indicated that highly glycosylated gp120 did not introduce any constraints on the CTLA4 portion having the propensity to interfere with its binding to B7 molecules. Although the mechanisms of immune enhancement mediated by CTLA4:gp120 are not clearly understood, it is likely that the B7-binding property of the fusion protein could play a critical role in the process of T-cell activation. Our observation is consistent with previous studies based on the CTLA4 fusion approach (11, 22) in which a DNA vaccine encoding CTLA4: huigg was shown to induce high levels of antibodies (11) and directed delivery of antigens to the site of immune induction (lymph nodes) is a crucial factor to elicit antiviral immunity in mice (22). Our study confirms and extends this novel approach to the complex HIV-1 envelope glycoprotein. We have not tested the ability of the vaccine-induced anti-gp120 antibodies in viral neutralization assays in vitro. In this study, we mainly focused on the longitudinal analysis of gp120-specific CD8 T cells and antibody responses within individual mice. This has provided us the opportunity to assess the generation and subsequent evolution of these responses in this murine model system. In the case of antigen-specific CD8 T cells, we assessed their ability to produce IFN-, a surrogate marker for functional CD8 T-cell activity in various models of infection and immunization (2, 6, 7, 9, 32, 48, 49). There are two distinct modes of activating antigen-specific CD8 T cells (23, 28, 52). One mode involves processing of the antigen by intracellular machinery (ubiquitin-proteosome pathway, ER) so that MHC class I-peptide complexes could be recognized by T-cell receptors with different specificities on T cells (endogenous pathway). The other mode involves delivery of antigen from the extracellular space by endocytic mechanisms to access the cytosol of APC (exogenous pathway crosspriming). The biosynthesis and maturation of HIV-1 or other viral envelope glycoproteins occur in the mammalian secretory pathway, thereby restricting easy access to the machineries of immune induction in APC (27, 38, 51, 71). In the case of DNA vaccines, it appears that both pathways could operate to induce CD8 T-cell responses (28, 52). Antigen-specific activation of T cells occurs in the secondary lymphoid organs in response to the sampling of antigen-loaded DCs that migrate from the peripheral tissue. We have demonstrated that both gp120 and sctla4:gp120 are secreted from mammalian cells; therefore, both should be taken up by APC with similar efficiencies for antigen processing and subsequent elicitation of immune responses, especially antibody responses. However, only the fusion protein, CTLA4:gp120, consistently showed enhanced CD8 T-cell and antibody responses, suggesting that this protein was endowed with the capacity to efficiently utilize either the endogenous or exogenous pathway or both (23, 28, 52). In natural HIV-1 infections, the presence of Env-specific CD8 T cells (cytotoxic T lymphocytes) is correlated with a reduction in viremia, indicating their importance in early viral control in HIV-infected individuals (9, 29, 43, 50). However, the complexities inherent in the structural domains of the HIV-1 envelope glycoproteins pose special challenges in the induction of a robust neutralizing antibody response in a number of vaccine strategies (2, 3, 14 16, 26, 32, 46, 74, 75). We have shown here that the modulation of immune responses by the fusion protein is dependent on the delivery sites of immunization. Injection of the DNA vaccines by the i.m. route induced only modest levels of gp120-specific CD8 T cells with relatively high antibody titers. On the other hand, a s.c. booster injection given after a primary i.m. immunization but before another booster injection through the i.m. route elicited the highest levels (11%) of gp120-specific CD8 T cells. This mode of DNA vaccination appears to be more efficient in the induction of antigen-specific CD8 T cells by both gp120 (3.5%) and CTLA4:gp120 (11%) DNA. Surprisingly, an s.c. booster injection given after the i.m. primary immunization did not show any enhancement in the levels of antigen-specific CD8 T cells until the mice were given a second booster injection via the i.m. route. Importantly, there is a concomitant increase in the anti-gp120 antibody titers by this vaccination protocol as well. The quality of the antibody response (Th1 versus Th2) appears to be consistent with the route of immunization for both gp120 and CTLA4:gp120 DNA, indicating that covalent modification of gp120 with CTLA4 did not alter this aspect of the antibody response (11, 25). Thus, both the humoral and cellular arms of the immune system appear to be activated in a temporal fashion in response to the DNA vaccines expressing gp120 and CTLA4:gp120 delivered into two separate immunization sites. We do not fully understand the mechanisms by which this mode of vaccination activates the immune system, eliciting high levels of antigen-specific CD8 T cells and antibodies. This activation probably reflects the type or abundance of APC capable of processing gp120 and its variant form, CTLA4: gp120, at the tissue sites of antigen delivery. This is clearly evident from the experiments where g.g. immunization induced a robust antibody response with only moderately high levels of antigen-specific CD8 T cells. Many of the DNA vaccine studies are inherently inadequate in providing the precise mechanisms of immune induction, largely because of the complexity of APC biology (DC biology) in various tissue sites (56, 57, 69), as well as the multitude of different antigens being tested for vaccine efficacy (31). However, we and others have provided evidence that viral envelope glycoproteins could be covalently modified to enhance their immunogenicity in murine model systems (10, 22, 64, 65, 66; this study). Recently, impressive results have been obtained in the use of DNA vaccines as priming immunogens capable of effective containment of HIV replication in rhesus macaques (2, 6). This level of containment was achieved by boosting the immune responses of DNA-primed animals by recombinant modified vaccinia virus Ankara or cytokine (interleukin-2)-augmented vaccine modalities. Because of the simplicity of DNA vaccines in terms of their preparation and storage, it is a very attractive strategy for the induction of primary T-cell and antibody (Bcell) immune responses in animals. However, this mode of vaccination has limitations and elicits only modest levels of antigen-specific immune cells. Improving the efficacy of DNA vaccines as priming immunogens would tremendously help achieve the highly effective means of boosting the immune response by various vector systems. Such approaches have

10860 NAYAK ET AL. J. VIROL. clear advances in eliciting antiviral immune responses against HIV/AIDS. It would be of much interest to test the CTLA4-based DNA vaccine approach in large-animal models (for example, nonhuman primates) for efficacy trials. However, there could be important safety issues in the introduction of a DNA vaccine encoding a cellular protein as part of an immunogen. Further improvement in the refinement (with minimal cellular sequences attached to antigen) and delivery of antigens have a clear potential in the utility of this antigen-targeting approach in DNA vaccination protocols in the future. ACKNOWLEDGMENTS We thank Chris Ibegbu, Ashley Carter, and Mike Hulsey of the Emory University CFAR Immunology Core for help with flow cytometric analysis, and we thank S. Nagarajan of the Pathology Department, Emory University, for help with the experiments presented in Fig. 2. We also thank Rafi Ahmed for support. 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