Th1-biased immune responses induced by DNA-based immunizations are mediated via action on professional antigen-presenting cells to up-regulate IL-12 production

Abstract

The efficacy of DNA-based immunization in conferring protective immunity against certain microbial pathogens including human immunodeficiency virus type 1 (HIV-1) has been described. The potential advantage of DNA-based immunization over the traditional vaccines largely results from its capacity to efficiently induce Th1-biased immune responses against an encoded antigen. We describe how Th1-biased immune responses are induced by DNA-based immunization, using a DNA vaccine construct encoding HIV-1 gp160 cDNA and an eukaryotic expression plasmid carrying murine IFN-gamma cDNA. Transfection of an eukaryotic expression plasmid carrying immunostimulatory sequences (ISS) as well as a gene of interest (DNA vaccine) into professional antigen presenting cells (APC) induced transactivation of IL-12 mRNA, which resulted in antigen-specific Th1-biased immune responses against the encoded antigen. Th1-biased immune responses induced by DNA-based immunization were substantially upregulated by a codelivery of an ectopic IFN-gamma expression system, and this augmentation was mediated via action on professional antigen presenting cells to upregulate IL-12 production. Taken together, it appears likely that Th1-biased immune responses induced by DNA-based immunization are mediated via action on professional antigen-presenting cells to produce IL-12. Interestingly, the model provided strikingly resembles that previously described in infection with Listeria monocytogenes, an intracellular Gram-positive bacterium that induces strong Th1-biased immune responses. The result suggests that DNA-based immunization mimics certain aspects of natural infection with microbial organisms like attenuated vaccines, which in turn provides a rationale to the question of why DNA-based immunization so efficiently induces protective immunity against these microbial pathogens.

Fig. 1

Eukaryotic expression plasmid pBC12/CMV carrying HIV-1 gp160 cDNA, its restriction enzyme map and CpG motifs. Twenty CpG motifs are identified. The full sequence of pBC12/CMV carrying HIV-1 gp160 can be obtained through Y.A. or J.F. upon request. ‘CpG motifs’ without a palindrome sequence are indicated by asterisks.

Fig. 2

Peritoneal macrophhages (PECs) were isolated as described [45,46], and transfected in vitro with the DNA vaccine construct, IFN-γ expresion plasmid, IL-12 expression plasmid, or an empty expression plasmid using a calcium phosphate coprecipitation method. Twelve hours after transfection, total RNA was extracted. (a) One μg of total RNA was reverse-transcribed using AMV-reverse transcriptase and oligo(dT) as the primer, and then subjected to PCR reaction using Taq polymerase and the primers specific for murine IL-12 p40 gene. Left: Total RNA extracted from C3H/HeJ mice. Right: Total RNA extracted from BALB/c mice. (b) The same amount of RNA was subjected to RT-PCR reaction using the primers specific for murine IFN-γ gene. Conditions for RT-PCR reaction are described in detail in Materials and Methods section. ‘None’ indicates PECs without a transfection procedure.

Fig. 3

(a) Muscle tissues injected with 20 μg of IFN-γ expression plasmid together with the DNA vaccine construct were ressected 4 days after the injection, stained with anti-H2 IA^d antibody, and were visualized by FITC. Muscle fibres incorporating the plasmid DNA expressed IA^d molecules on the cell surfaces. (b) Muscle tissues injected with 2μg of the DNA vaccine construct alone. Muscle tissues were ressected 4 days after the injection and stained with anti-H2 IA^d antibody.Only the vascular tissues, but not the muscle fibres, were stained with anti-H2 IA^d antibody.

Fig. 4

(a) IgG titres against recombinant gp160 protein. Serum samples were obtained 4 weeks after the primary immunization and HIV-1 gp160 specific IgG titres were titrated out using the preimmune samples. IIIB alone, HIV-1 gp160 specific IgG titres from the groups of mice (a result from one of two independent series of experiments, n = 5–10 for each group for each experiment) treated with the DNA vaccine construct alone; IFN-10, HIV-1 gp160 specific IgG titres from the groups (the result from the same experiment) of mice which received 10 μg of IFN-γ expression plasmid together with the DNA vaccine construct; IFN-20, groups (the result from the same experiment) of mice which received 20 μg of IFN-γ expression plasmid together with 2 μg of the DNA vaccine construct; anti-10, groups (the result from the same experiment) of mice which received 10 μg of IFN-γ expression plasmid as well as the DNA vaccine construct and treated with anti-IFN-γ neutralizing monoclonal antibody, XMG1.2; anti-20, groups (the result from the same experiment) of mice which received 20 μg of IFN-γ expression plasmid as well as the DNA vaccine construct and treated with anti-IFN-γ neutralizing monoclonal antibody, XMG1.2; empty, groups (the result from the same experiment) of mice which received empty expression plasmid. Results are expressed by box-and-whisker plots. Mann–Whitney U-test was used for statistical analysis; n = 5 for each group. (b) Using a two step ELISA system, antigen-specific immunoglobulin subclass profiles were characterized. Serum samples obtained 4 weeks after the primary immunization were diluted 1:50, and antigen-specifc immunoglobulin subclasses were detected by incubating the wells with goat-antimouse subclass specific immunoglobulin (Sigma). Wells were then treated with HRP-conjugated rat antigoat antibody and colour reactions were developed by O-phenylenediamine dihydrochloride. Absorbance values from antimouse IgG1 specific antibody were divided by those from antimouse IgG2a specific antibody. Results are expressed by box-and-whisker plots (n = 5–10; *statistically significant; Mann–Whitney U-test; n = 5 for each group).

Fig. 5

Cytokine production profiles of the splenocytes restimulated in vitro for 48 h with HIV-1 gp160 V3 peptide which carries a T helper cell epitope. Cytokine profiles were assesed based on the protocols originally described by Pertmer et al. [27]. Groups of mice (results from two indenpendent series of experiments; n = 18 for each group, P = 0.048 < 0.05*; Mann–Whitney U-test) were subjected to assays approximately 4–5 weeks after the primary immunization. Abbreviations used in this figure are described in the legend to Figure 4. Mean ± SE values are indicated. (a) IFN-γ production; (b) IL-4 production. *Statistically significant.

Fig. 6

CTL responses against the target cells pulsed with HIV-1 gp160 specific H2D^d-restricted cytotoxic T cell epitope peptide (RGPGRAFVT). Groups of mice (the result from one of three independent series of experiments, n = 4–10 for each group for an experiment) were subjected to CTL assays approximately 4 weeks after the primary immunization. For grouping of mice, see the legend to Figure 4. CTL activity against the target cells not pulsed with the same peptide was simultaneously assessed and all the results were less than 5% specific lysis. Effector to target ratios were 80:1, 40:1, and 20:1, respectively. Mean ± SE values are shown. *Statistically significant; Mann–Whitney U-test, n = 4, 6; at an effector to target ratio of 80 : 1.