Comparison of immune responses generated by optimized DNA vaccination against SIV antigens in mice and macaques

Abstract

Optimized DNA vectors were constructed comprising the proteome of SIV including the structural, enzymatic, regulatory, and accessory proteins. In addition to native antigens as produced by the virus, fusion proteins and modified antigens with altered secretion, cellular localization and stability characteristics were generated. The DNA vectors were tested for expression upon transfection in human cells. In addition, the vectors were tested either alone or in combinations in mice and macaques, which provided an opportunity to compare immune responses in two animal models. DNA only immunization using intramuscular injection in the absence or presence of in vivo electroporation did not alter the phenotype of the induced T cell responses in mice. Although several fusion proteins induced immune responses to all the components of a polyprotein, we noted fusion proteins that abrogated immune response to some of the components. Since the expression levels of such fusion proteins were not affected, these data suggest that the immune recognition of certain components was altered by the fusion. Testing different DNA vectors in mice and macaques revealed that a combination of DNAs producing different forms of the same antigen generated more balanced immune responses, a desirable feature for an optimal AIDS vaccine.

Fig. 1

Optimization of SIV Gag expression and immunogenicity in mice. (A) Human 293 cells were transfected in triplicate with 50 ng of gag expression plasmids 1S and 206S and 100 ng of the GFP expression plasmid FRED143. Two days later the cells were harvested and the Gag and GFP levels were measured. Total p27gag (cell-associated and supernatant) and relative GFP values (×100) and SEM are shown. Expression of p57gag from plasmid 206S resulted in 1.5× higher Gag production compared to the levels obtained by plasmid 1S. (B) Comparison of expression of p57gag from the CMV.kan (plasmid 206S) and pVAX plasmids. The two plasmids contain the same p57gag gene including the optimized AUG initiator sequence. HEK 293 cells were transfected with 50 ng of the indicated plasmids and 2 days later Gag expression was measured by antigen capture assay. The mean and SD of triplicate transfection are shown. The mean GFP values (×100) obtained from the cotransfected pFRED143 plasmid are 657 and 740 for p57gag/CMV.kan and p57gag/pVAX, respectively. (C) The cartoon depicts the native p57gag and p39gag and modifications thereof. The fusion proteins comprise the human MCP-3 and p57gag or the p39gag; the murine MCP-3 and p39gag. Plasmids expressing the different forms of Gag were transfected into human 293 cells. The expression vectors producing the native p57gag (plasmid 1S), the fusion of MCP-3 with p57gag (plasmid 214S), p39gag (plasmid 208S), the fusions of human MCP-3 (plasmid 209S) and the murine MCP-3 (plasmid 213S) with p39gag are shown. Human 293 cells were transfected with the p57gag plasmid series or the p39gag plasmid series, respectively, in different experiments. Two days later, the cells were harvested. The Gag proteins from the supernatants (1/250 of sample) and the cell extracts (1/250 of sample) were separated on SDS-PAGE and were visualized on Western immunoblot using pooled antiserum from SIVmac251 infected macaques. Transfection efficiencies were controlled by co-transfection of GFP encoding plasmid pFRED143. The relative GFP values (×100) were 260 and 150 for the p57 plasmid series, and 220, 170 and 160 for the p39 plasmid series respectively. (D) Groups of Balb/c mice (N=6) were immunized with DNA plasmids producing the native p57gag (plasmid 1S) or the murine MCP3-p39gag (plasmid 171S) at day 0 and week 4 and were sacrificed 2 weeks later. Gag-specific cellular immune responses were analyzed in pooled splenocytes by flow cytometric analysis. (E) The endpoint titers of the Gag-specific binding antibody from the mice that received the plasmids described in panel B were determined by ELISA from individual serial 4-fold diluted plasma samples. Statistical analysis was performed using two-tailed Mann Whitney t test.

Fig. 2

Immunogenicity of native and modified SIV Env proteins in mice. (A) The cartoon depicts the native gp160env as well as the modified gp160env proteins which have the native signal peptide replaced by the human or murine MCP-3 chemokine or the tPA signal peptide. Human 293 cells were transfected with 100 ng of the plasmids expressing the native gp160env (plasmid 64S, lane 1; plasmid 99S, lane 2); the modified gp160env proteins with the native signal peptide replaced by the human (plasmid 73S, lane 3) or murine MCP-3 (plasmid 115S, lane 5) chemokine or the tPA signal peptide (plasmid 78S; lane 4). Two days later, the cells were harvested and 1/250 of the supernatants and cell extracts, respectively, were visualized by Western immunoblot analysis using pooled antiserum from SIVmac251 infected macaques. Transfection efficiency was controlled by co-transfection of GFP encoding plasmid pFRED143. The relative GFP values (× 100) obtained from lanes 1-5 were: 30, 40, 110, 100 and 50, respectively. (B) Immunogenicity of Env produced from the indicated plasmids was assessed. Groups of Balb/C mice (N=5-6) received the plasmids expressing the native (plasmid 64S), the human MCP3-env (plasmid 73S) or murine MCP3-env (plasmid 115S) or the tPA-env (plasmid 78S). The mice were vaccinated 3 times (day 0, week 3 and week 6) and were sacrificed 2 weeks later. Env-specific cellular immune responses were analyzed from splenocytes of individual mice and provided as mean and standard error (SEM) of IFN-γ-producing CD4⁺ and CD8⁺T cells. (C) The endpoint titers of the Env-specific binding antibody from the individual mice described in panel B were determined by ELISA from serial 4-fold dilutions of individual plasma samples. The mean titers are indicated.

Fig. 3

Immunogenicity of different mixtures of plasmids expressing Gag and Env in mice and macaques. (A) Groups of Balb/c mice (N=6) were immunized with a mixture of plasmids (50 μg each) expressing the native forms of p57gag (plasmid 1S) and gp160env (plasmid 99S) or a mixture of plasmids expressing the modified forms of Gag (25 μg of the CATE-p57gag [plasmid 2S] and 25 μg MCP3-p39gag [plasmid 21S]) and 50 μg MCP3-env (plasmid 73S). A third group of mice received a combination of the DNA plasmids (“combined antigens”) expressing the modified Gag (25 μg of each gag plasmid 2S and 21S) and native gp160env (50 μg of the env plasmid 99S). The mice were immunized at day 0 and week 4 and were sacrificed 2 weeks later. Gag- and Env-specific cellular immune responses were measured by ELISPOT in splenocytes from individual mice. The mean and SEM are shown. (B) Gag- and Env-specific endpoint titers of binding antibodies from pooled plasma samples from the mice shown in panel A were determined by ELISA and the log of antibody titers are shown and the mean is shown. (C) Indian rhesus macaques (N=8) were vaccinated with a mixture of plasmids expressing native or modified SIV antigens using IM delivery followed by in vivo electroporation. The data of the group receiving the native and modified forms of the antigens are modified from Rosati et al. [31] and show the data from week 4 post EP4. The mean and SEM are shown. (D) A group SIV-infected and ART-treated macaques (N=4) received a mixture of DNAs expressing thecombination of the native and modified antigens using IM delivery followed by in vivo electroporation. The cellular responses (mean and SEM) from the time of EP1 and 2 weeks post EP2 are shown. (E) Endpoint Env and Gag antibody titers were determined by ELISA from the animals (shown in panel C) by ELISA using individual plasma of macaques immunized with DNA expressing the native (left panel) and modified (right panel) antigens. Mean and SEM are shown.

Fig. 4

Increased immunogenicity by the modified SIV-pol. (A) The cartoon depicts the Pol protein that contains mutations inactivating the enzymatic activities in protease, reverse transcriptase, RNaseH, and integrase (plasmid 88S, lane 1). Fusions of Pol with LAMP (plasmid 103S, lane 2) or with murine MCP-3 that has the murine GM-CSF (plasmid 195S, lane 3) or murine IP-10 (plasmid 196S, lane 4) signal peptide are shown. The fusion of Pol to human MCP-3 with the murine IP-10 signal peptide is shown (plasmid 216S, lane 5). Human 293 cells were cotransfected with 100 ng of the indicated plasmids and 100 ng of GFP expressing plasmid, pFRED143. Gag protein expression was analyzed 2 days later by Western immunoblot using pooled plasma from SIVmac251 infected macaques. Data shown in lanes 1 and 2 and in lanes 3-5 are from two independent experiments. The relative GFP values (×100) were: lanes 1 and 2: 340, 220; lanes 3-5: 140, 160, and 140. (B) Balb/c mice (N=12) were immunized with different pol expression plasmids at day 0 and week 4 and sacrificed 2 weeks later. Splenocytes of individual animals were analyzed by ELIspot assay. Lane 1, pol (plasmid 88S); lane 2, LAMP-pol (plasmid 103S); lane3, muMCP3-pol with the murine GM-CSF leader (plasmid 195S); lane 4, muMCP3-pol with the murine IP-10 signal peptide (plasmid 196S). Statistical analysis was performed using one-way ANOVA, Dunn's Multiple comparison test. The mean values are indicated. (C) Pooled splenocytes were analyzed by flow cytometric analysis and the levels of Pol-specific IFN-g⁺ CD4⁺ and CD8⁺ T cells were measured. (D) Pooled plasma from the mice immunized with Pol (panel B, lane 1), LAMP-pol (panel B, lane 2) and muMCP3-pol (panel B, lane 4) were used in a Western immunoblot assay to detect Pol proteins produced upon transient transfection of human 293 cells with 1 μg of the Pol plasmid (plasmid 88S). Two days later cell extracts were harvested in 0.5X RIPA and loaded on to a 12% NuPAGE Bis-Tris gel. The blot was probed using a 1:200 dilution of pooled plasma samples from the 3 vaccinated groups of mice. (E) The rhesus macaques described in Fig. 3C were coimmunized with a plasmid expressing the native or the modified Pol proteins as indicated. The immune responses determined by flow cytometry were measured at 14 weeks post EP4. The percent of pol-specific IFN-g producing CD4⁺ and CD8⁺ T cells are shown (mean and SEM). (F) The rhesus macaques described in Fig. 3D were co-immunized with the plasmid expressing LAMP-Pol. The immune responses determined by flow cytometry were measured at day of EP1 and 2 weeks post EP2. The percent of pol-specific IFN-g producing CD4⁺ and CD8⁺ T cells are shown (mean and SEM) as described for panel E.

Fig. 5

Immunogenicity to accessory proteins. (A) Cartoon depicts fusion proteins consisting of Rev (aa 1-107), Nef (aa 4-260) and Tat (aa 2-130). Two configurations changing the position of Nef (plasmid RevNefTat [RNT, 166S] and RevTatNef [RTN, plasmid 168S]; and the b-Catenin-RevNefTat (CATE-RNT; RNT [plasmid 174S] are shown. Expression of the proteins from 293 cells transfected with 100 ng of the respective DNAs was visualized on Western immunoblots probed with anti-Nef antibody. The relative GFP relative values (×100) of lanes 1-3 were: 300, 270, and 300, respectively. (B) Immunogenicity in Balb/c mice upon IM vaccination using plasmids expressing the fusion proteins shown in panel A. Mice were immunized at day 0 and week 4 with 100 μg of the indicated plasmids and sacrificed 2 weeks later. The levels of Rev-, Nef- and Tat-specific cellular immune responses were determined by ELISPOT assay. The mean and SEM are shown. (C) Cartoon depicts fusions of NefTat with LAMP and Vif Expression of the proteins from 293 cells transfected with 200 ng of DNA was visualized on Western immunoblots probed with anti-Nef antibody. The relative GFP values (×100) of the transfection in lanes 1-4: 240, 250, 270, and 260, respectively. (D) Immunogenicity of the NTV and LAMP-NTV fusion proteins shown in panel C. Balb/C mice (N=6) were immunized at day 0 week 3 and week 6 with 100 μg of the indicated plasmids and sacrificed 2 weeks later. Cellular immune responses to Nef, Tat and Vif were determined from splenocytes from individual mice (left panel) and antigen-specific CD4+ and CD8+ T cells were measured (right panels). Statistical analysis was performed using two-tailed Mann Whitney t test, with p values of 0.043 (Nef), 0.043 (Tat) and 0.0022 (Vif). The mean and SEM are shown. (E) The humoral immune responses to Nef were analyzed from plasma samples from individual mice (right panel) as endpoint ELISA titers. The mean is shown. (F) Immunogenicity of NTV and LAMP-NTV in rhesus macaques. Naïve macaques (see Figure 3C) were vaccinated with NTV and LAMP-NTV plasmids as described [31]. The levels of antigen-specific IFN-g producing total (left panel) and CD4+ and CD8+ T cells (right panels) were measured by flow cytometry at 14 weeks post EP4. Mean and SEM are shown. (G) The ART-treated macaques were co-immunized with the LAMP-pol expression vector. The immune responses determined by flow cytometry were measured at the day of EP1 and 2 weeks post EP2. The percent of antigen-specific IFN- g producing CD4⁺ and CD8⁺ T cells are shown (Mean and SEM).

Fig. 6

Loss of Nef Immunogenicity when Nef is expressed as part of GagPol or Pol fusion proteins. (A) The Pol-NTV (plasmid 35S) and the CATE-Pol-NTV (plasmid 44S) fusion plasmids were expressed in human 293 cells upon transfection of 100 ng of DNA. Two days later the cell extracts were analyzed on Western immunoblot using pooled plasma from SIVmac251 infected macaques. The relative GFP relative values (× 100) shown in lanes 1-2 were 570 and 570, respectively. (B) The immunogenicity of the pol-NTV fusion proteins. Balb/c mice (N=6) were vaccinated twice with 100 μg of DNA at day 0 and week 4, and splenocytes were prepared 2 weeks later. Cellular immune responses to pol, Nef, Tat, and Vif peptide pools were analyzed by ELISPOT. The mean and SEM are shown. (C) The GagPol fusion protein contains Gag (aa 1-439) artificially linked (via 13 aa of the ORF preceding Pol) to Pol (aa 1-951) (plasmid 82S). Nef was fused to the C-term (plasmid 179S) or the N-term (plasmid 185S) of GagPol. The plasmids expressing the fusion proteins were expressed in human 293 cells and analyzed on Western immunoblot probed with pooled plasma from SIVmac251 infected macaques. Plasmids expressing only Gag (plasmid 10S; non-myristoylated gag), Pol (plasmid 88S) and Nef (plasmid 180S) are shown. The relative GFP values (× 100) of the transfections shown in lanes 1-6 were: 140, 150, 150, 150, 150 and 140. (D) The immunogenicity of the indicated GagPol fusion proteins was tested. Balb/c mice (N=6) were vaccinated with 100 μg twice at day 0 and week 4, and splenocytes were prepared 2 weeks later. Cellular immune responses to Gag, Pol, and Nef peptide pools were analyzed by ELISPOT. The mean and SEM are shown.