Immunostimulatory DNA as an adjuvant in vaccination against Leishmania major - PubMed (original) (raw)

Immunostimulatory DNA as an adjuvant in vaccination against Leishmania major

K J Stacey et al. Infect Immun. 1999 Aug.

Abstract

Oligodeoxynucleotides (ODN) which contain immunostimulatory CG motifs (CpG ODN) can promote T helper 1 (Th1) responses, an adjuvant activity that is desirable for vaccination against leishmaniasis. To test this, susceptible BALB/c mice were vaccinated with soluble leishmanial antigen (SLA) with or without CpG ODN as adjuvant and then challenged with Leishmania major metacyclic promastigotes. CpG ODN alone gave partial protection when injected up to 5 weeks prior to infection, and longer if the ODN was bound to alum. To demonstrate an antigen-specific adjuvant effect, a minimum of 6 weeks between vaccination and infection was required. Subcutaneous administration of SLA alone, SLA plus alum, or SLA plus non-CpG ODN resulted in exacerbated disease compared to unvaccinated mice. Mice receiving SLA plus CpG ODN showed a highly significant (P < 5 x 10(-5)) reduction in swelling compared to SLA-vaccinated mice and enhanced survival compared to unvaccinated mice. The modulation of the response to SLA by CpG ODN was maintained even when mice were infected 6 months after vaccination. CpG ODN was not an effective adjuvant for antibody production in response to SLA unless given together with alum, when it promoted production of immunoglobulin G2a, a Th1-associated isotype. Our results suggest that with an appropriate antigen, CpG ODN would provide a stable, cost-effective adjuvant for use in vaccination against leishmaniasis.

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Figures

FIG. 1

Lesion sizes in vaccinated mice challenged with L. major. BALB/c mice were injected with indicated vaccine preparations in the left footpad, followed 2 weeks later by injection s.c. in the rump; 5 weeks later, these mice and resistant CBA/Ca mice were infected in the right footpad with 2 × 106 L. major promastigotes, and footpad swelling was monitored weekly. The mean and standard error of measurements from groups of five or six (control group only) mice are shown up to the time when the first mouse from that group had to be killed. Treatments: □, SLA plus CpG ODN (AAC); ◊, SLA plus non-CpG ODN; ○, CpG ODN; ▵, SLA; ■, control unvaccinated mice; ⧫, resistant CBA/Ca mice.

FIG. 2

Measurement of nitrite as an indication of NO produced by RAW264 cells in response to DNA. Cells were incubated with IFN-γ (40 U/ml) for 1 h before various concentrations of DNA were added for 24 h. DNAs used: □, DNA from M. lysodeikticus; ◊, CpG ODN (AAC); ▵, non-CpG ODN (AAG). Results are the average of nitrite concentration in triplicate wells.

FIG. 3

Lesion sizes and survival of vaccinated mice challenged with L. major. BALB/c mice were injected in the left footpad with the indicated vaccine preparations, followed 2 weeks later by injection s.c. in the rump; 6.3 weeks later, these mice and resistant CBA/Ca were infected in the right footpad with 2 × 106 L. major promastigotes. All groups contained five mice except for control (six mice) SLA-CpG ODN (10 mice), and CpG ODN-alone (12 mice) groups. Results from groups of mice treated with either of the CpG ODNs AAC and AO-1 were indistinguishable and were combined to give SLA-CpG ODN and CpG ODN groups in order to simplify the graphs and improve statistical power. For footpad measurements, results show the mean and standard error up to the time when the first mouse had to be killed. Mice were killed according to Home Office guidelines when lesions began to ulcerate. (A) Footpad sizes of infected mice in the weeks following infection. Treatments: □, SLA plus CpG ODN; ○, CpG ODN; ▵, SLA; ■, control unvaccinated mice; ⧫, resistant CBA/Ca mice. (B) Survival of infected animals. Treatments were as for panel A. (C) Footpad sizes of mice receiving vaccinations which exacerbated infection. Treatments: ▵, SLA; ◊, SLA plus non-CpG ODN; ⧫, SLA plus micrococcal DNA; □, SLA plus alum; ●, SLA plus alum and micrococcal DNA; ○, SLA plus IL-12; ■, control unvaccinated mice. (D) Footpad sizes of mice treated with SLA plus alum (□) or SLA plus alum and CpG ODN (AAC) (▵) or alum and CpG ODN (AAC) (○). (E) Survival of mice treated as for panel D and of control unvaccinated mice (■).

FIG. 4

Lesion sizes in mice challenged with L. major at 6 weeks and 6 months after vaccination. Treatments: ▵, SLA; □, SLA plus CpG ODN (AAC). Results shown are the mean and standard error of footpad measurements. (A) Groups of three BALB/c mice were vaccinated as for Fig. 3. Six months later, they were infected in the right footpad with 2 × 106 L. major promastigotes, and footpad size was monitored in the weeks following infection. (B) Groups of 9 (SLA plus CpG ODN) or 10 (SLA) BALB/c mice were vaccinated twice s.c. in the rump at a 2-week interval; 6.5 weeks later, they were infected in the right footpad at the same time as the mice used for panel A.

FIG. 5

Relative anti-SLA IgG1 and IgG2a levels in vaccinated mice from two experiments (i and ii). Mice were bled 4 weeks after the second vaccination. Antibody levels are expressed relative to a standard pool (see Materials and Methods). Mean and standard error of Ig levels measured in duplicate on at least five animals per group are shown. DNAs used are CpG ODNs (AAC and AO-1), non-CpG ODN (AAG), and micrococcal DNA (mDNA).

FIG. 6

Relative anti-SLA IgG1 and IgG2a levels in infected mice from the experiment in Fig. 3. Mice were bled 2.5 weeks after infection. Results shown are mean and standard error of Ig levels measured in duplicate on at least five animals per group. DNAs used were CpG ODNs (AAC and AO-1) and non-CpG ODN (AAG). (A) Antibody levels expressed relative to a standard pool (see Materials and Methods). (B) Ratio of anti-SLA IgG1 to IgG2a.

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Immunostimulatory DNA as an adjuvant in vaccination against Leishmania major - PubMed (original) (raw)