Adjuvants may reduce in vivo transfection levels for DNA vaccination in mice leading to reduced antigen-specific CD8+ T cell responses

Immunization with piggyBac DNA vaccines in PBS induces stronger antigen expression than DNA vaccination using cationic liposomes, Adjuplex, or JetPEI

In these studies pmhyGENIE-3 piggyBac vectors that catalyze the insertion of a transgene-containing transposon were used to vaccinate mice and generate antigen-specific immune responses. The efficacy of these DNA vaccines may be improved by including adjuvants that promote in vivo transfection and immune system activation. We designed experiments to compare 3 adjuvants with different chemical structure: DOPE/DOTAP cationic liposomes, Adjuplex and JetPEI (Fig. 1). Mice were injected with piggyBac integrating DNA plasmids formulated with PBS or DNA plasmids in combination with one of the 3 different adjuvants. Unvaccinated mice were used as controls. Innate immune responses were evaluated in lymph nodes of vaccinated mice 72h after the first immunization using pmhyGENIE-3-eGFP. Adaptive eGFP-specific T cell responses were then evaluated in spleen cells isolated 10 days after boost vaccination with pmhyGENIE-3-eGFP. The presence of eGFP-specific CD8⁺ T cells was also evaluated in the blood of these mice using eGFP-specific pentamers.

The efficacy of the 3 adjuvants in promoting in vivo transfection and consequent antigen production was evaluated by measuring luciferase activity 24h after the second vaccination with pmhyGENIE-3-GL3 (Fig. 2). This luciferase encoding plasmid was used since in vivo protein expression is quantifiable using the IVIS imaging system that detects in vivo luciferase activity. Formulations of piggyBac plasmids diluted in PBS or combined with either liposomes, Adjuplex, or JetPEI were s.c. injected in the hind flank of mice. The day after the second vaccination a significantly higher activity of luciferase was detected in mice injected with DNA suspended in PBS. Plasmid inoculation using Adjuplex or JetPEI also induced significant luciferase expression, although at lower levels compared to DNA in PBS. Within the group of mice vaccinated using piggyBac plasmids in conjunction with liposomes, levels of transgene expression was not equal. IVIS imaging of luciferase activity showed that some mice exhibited a detectable luciferase signal while others did not (Fig. 2B). These data suggest that plasmid DNA diluted in PBS may be taken up by host cells following s.c. injection and produce considerable amounts of antigen. In contract, all 3 adjuvants interfered with this process as indicated by lower levels of luciferase activity produced after vaccination.

Adjuvants generate innate immune responses with activation of dendritic cells in lymph nodes of vaccinated mice

It has been demonstrated that adjuvants trigger innate immune responses and these responses are central to their adjuvant activity.¹⁴ Since innate responses typically precede adaptive responses, the effects of liposomes, Adjuplex and JetPEI were evaluated 3d after vaccination. Cells isolated from draining lymph nodes of vaccinated mice were enumerated and populations of immune cells identified using flow cytometry analysis. Results from mice vaccinated using the different adjuvants were compared with those from mice vaccinated without adjuvants and unvaccinated mice. A significantly larger number of cells were found after vaccination with liposomes or Adjuplex, compared with controls (Fig. 3A). Vaccination using JetPEI and non-adjuvant DNA did not increase lymph node cell numbers. Percentages of the different leukocyte populations were assessed using multicolor flow cytometer analysis, which allows the simultaneous identification of several different cell types in one sample.¹⁵ We identified surface markers for different types of leukocytes including T cells (CD3, CD4 and CD8), B cells (B220), granulocytes (Gr-1), monocytes/macrophages (CD11b) and dendritic cells (CD11c). Our data show that liposomes caused an increase in the relative abundance of cells involved in the innate immune response. In particular, a higher abundance of B cells, granulocytes and macrophages were found in mice injected with liposomes compared with unvaccinated mice. Mice vaccinated using Adjuplex exhibited a higher abundance of granulocytes and macrophages, while mice vaccinated using JetPEI and non-adjuvant vaccinated mice only exhibited increased abundance for macrophages (Fig. 3B, C, D).

We also analyzed cells involved in the adaptive immune response. Dendritic cells, CD4⁺ T cells and CD8⁺ T cells did not increase in relative abundance after vaccination with the different conditions, except in the case of Adjuplex that caused reduced levels of dendritic cells compared with controls (Fig. 3E, F, G). Analysis of dendritic cell activation markers, CD40 and CD80, indicated that vaccination using any of the 3 adjuvants promoted the maturation of dendritic cells as indicated by levels of these 2 co-receptors. In contrast, mice vaccinated without adjuvants showed the same levels of activated dendritic cells than unvaccinated mice (Fig. 3H, I).

Only DNA injection in PBS or formulated with adjuplex generates detectable antigen-specific CD8⁺ T cells in blood of vaccinated mice

Since eGFP represents a non-self protein that can serve as a vaccine antigen for mice, we utilized the plasmids encoding this protein to compare vaccine adjuvants. Pentamer reagents are available that detect mouse CD8⁺ T cells specific for the eGFP 200–208 epitope, which were used to evaluate eGFP specific CD8⁺ T cells generated in vaccinated mice. Results showed that, after both the primary and secondary vaccination with pmhyGENIE-3-eGFP in PBS or in combination with Adjuplex, significantly more eGFP-specific CD8⁺ T cells were detected in the blood of these mice compared to unvaccinated controls (Fig. 4). In contrast, DNA vaccination using liposomes or JetPEI did not produce significantly higher eGFP-specific CD8⁺ T cells in the blood.

Vaccination with piggyBac DNA in combination with liposomes or Adjuplex generates functional CD8⁺ T cells that produce IFNγ

We next investigated whether the higher numbers of CD8⁺ T cells from mice vaccinated without adjuvants or with Adjuplex corresponded to increased function of these cells. Different groups of mice were given primary and secondary s.c. injections in the hind flank with 40 μg/mouse of integrating plasmid formulated with the different adjuvants as described in the Materials and Methods section. Conventional re-stimulation assays were used to test functionality of CD8⁺ T cells. Specifically, spleens were removed from the eGFP plasmid DNA vaccinated mice on day 28 and splenocytes pulsed with eGFP peptide (200–208 epitope). Intracellular staining and flow cytometry analyses were used to measure the level of IFNγ within the CD8⁺ T cells. For all of the DNA formulations, with or without adjuvants, results showed significantly higher numbers of IFNγ producing splenocytes after stimulation with eGFP peptide (Fig. 5). Interestingly, the strongest IFNγ responses in CD8⁺ T cells were in mice vaccinated without adjuvants.

Mice

Female BALB/cJ mice at 4–8 weeks of age (Jackson Immunolabs, #000651) were maintained in a specific pathogen-free animal facility for at least 1 week before experiments. Procedures were performed in accordance with institutional guidelines approved by University of Hawaii Institutional Animal Care and Use Committee (IACUC).

Vaccination procedures

In experiments to evaluate antigen expression, mice were injected s.c. with 40 μg of piggyBac integrating plasmid DNA expressing luciferase, pmhyGENIE-3-GL3. In other experiments, analyses of immune responses were conducted in mice injected with a piggyBac integrating plasmid DNA expressing eGFP, pmhyGENIE-3-eGFP.^3,20 Each mouse was immunized with plasmid DNA diluted in 100μl of PBS or formulated in 100μl of DOTAP/DOPE liposomes (Avanti Polar Lipids, #850725 and #890890) or 100μl of adjuvants Adjuplex (Sigma-Aldrich, #A0362) or in vivo-JetPEI (Polyplus transfection, # 201).

Adjuvants

Liposomes containing DOTAP and DOPE (Avanti Polar Lipids, Alabaster, AL, USA) in a 1:1 molar ratio were prepared by drying the lipid mixture in chloroform under a stream of nitrogen as a thin layer in a 10-mL round-bottomed tube. The lipid film was hydrated in PBS to give a final concentration of 10 mg lipids/mL and then sonicated with Avanti sonicator. The lipid suspension (100 μl/mouse) was mixed with DNA (40 μg/mouse) and incubated for 10 min before injection into mice. For each mouse vaccinated using Adjuplex, 40 μg of DNA in 80 μl of PBS were mixed to 20 μl of adjuvant, 10 min before injection. JetPEI vaccination was formulated by mixing together 2 10% glucose solutions, one containing 40 μg of DNA and the other including 3 μl of in vivo-JetPEI. After mixing the 2 solutions, mice were injected within 10 min.

Measurement of antigen expression

Bioluminescent signals from luciferase-vaccinated mice were monitored using the IVIS Lumina (Perkin Elmer, Waltham, MA, USA) after i.p. injections of 0.1 mL of 15 mg/mL D-luciferin. Regions of interest were identified around the injection sites and quantified as total photon counts using Living Image™ software. The values were expressed as an average of all vaccinated mice for every treatment group.

Measurement of immune responses

Spleen cells of immunized mice (1 × 10⁶) were stimulated for 7 h with 10 μg/mL eGFP peptide (200-208 epitope) in the presence of 5 IU/mL IL-2 (BioLegend, #589102). For detection of IFN-γ in the cytoplasm, Golgi-plug (BD Biosciences, # 555029) was added 6 hours before staining. Fixation/permeabilization kits were used in combination with anti-CD3-PECy5, anti-CD8-APC, and anti-IFN-γ-PE (eBiosciences, #15-0031, #17-0081, #12-7311). Data were collected using the FACScalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Blood (200 μL) was collected from tails of BALB/c immunized mice. PE-labeled H2-Db pentamer (PENT) presenting the eGFP 200-208 epitope was purchased from Proimmune (#198) and cellular staining protocol performed as recommended by the manufacturer. Briefly, 2 × 10⁶ cells were stained with 10 μL of PENT at room temperature for 10 min, washed and further incubated for 20 min with anti-CD3-FITC, anti-CD8-APC and anti-CD16/32 PE-Cy5.5 (eBiosciences, #11-0032, #17-0081, #35-0161). PENT⁺/CD8⁺ cells were analyzed on a FACSCalibur Flow Cytometer and analyzed using FlowJo software. Inguinal lymph nodes were removed 72 h after vaccination and population of immune cells stained as described previously¹⁵ using FACScalibur flow cytometer. The following antibodies were used: anti-CD3-FITC, anti-CD4-PE/Cy7, anti-CD8-APC, anti-Gr1-PerCP/Cy5.5, anti-CD11c-PE, anti-B220-PE/TR and anti-CD11b-APC/Cy7 (BioLegend, #100306, #116016, #100712, #108427, eBiosciences #12-0114-81, BD Pharmigen #551489, #557657). CD3⁺ cells were previously gated before identification of CD4⁺ and CD8⁺ T cell populations. CD11c⁺ cells were previously gated before measuring their activation using antibodies anti- CD40-PE/Cy5 and anti-CD80-APC (eBiosciences #15-0401-81, # 17-0801-81).

Statistical analyses

All statistical tests were performed using GraphPad Prism (GraphPad 5.0). Means were compared using one-way ANOVA followed by the Bonferroni multiple comparison test. Statistical significance values are indicated in the figure legends.