Intralymphatic immunization enhances DNA vaccination

Abstract

Although DNA vaccines have been shown to elicit potent immune responses in animal models, initial clinical trials in humans have been disappointing, highlighting a need to optimize their immunogenicity. Naked DNA vaccines are usually administered either i.m. or intradermally. The current study shows that immunization with naked DNA by direct injection into a peripheral lymph node enhances immunogenicity by 100- to 1,000-fold, inducing strong and biologically relevant CD8(+) cytotoxic T lymphocyte responses. Because injection directly into a lymph node is a rapid and easy procedure in humans, these results have important clinical implications for DNA vaccination.

Figure 1

i.ln. immunization is the most efficient way to induce antiviral CTL responses. Groups of two C57BL/6 mice were immunized once with pEGFPL33A (0.02–200 μg) given i.d. or i.m. or i.spl. or i.ln. Positive control mice received 500 pfu LCMV i.v. Ten days after immunization spleen cells were isolated, and gp33-specific CTL activity was determined after secondary in vitro restimulation. Symbols represent individual mice; one of three similar experiments is shown.

Figure 2

i.ln. immunization is the most efficient way to induce antiviral anamnestic CTL responses. Groups of two C57BL/6 mice were immunized once with pEGFPL33A (0.2–200 μg) given i.d. or i.m. or i.spl. or i.ln. Positive control mice received 500 pfu LCMV i.v. Ten days after immunization mice were challenged with 5 × 10⁴ pfu LCMV i.v., and 4 days later spleen cells were isolated and ex vivo CTL activity was assayed. Symbols represent individual mice; one of two similar experiments is shown.

Figure 3

Repetitive i.m. immunization with pEGFPL33A plasmid DNA induces gp33-specific CTL. Groups of three C57BL/6 mice were immunized three times (on days 0, 7, and 14) with pEGFPL33A or with the control plasmid pEGFP-N3, given i.m. (200 μg per immunization) or i.spl. (20 μg per immunization) or i.ln. (20 μg per immunization). Seven days after the final immunization spleen cells were isolated, and gp33-specific CTL activity was determined after secondary in vitro restimulation. Positive control mice received 500 pfu LCMV i.v. Symbols represent individual mice; one of two similar experiments is shown.

Figure 4

i.ln. immunization elicits protective immunity against systemic and peripheral virus infection. (A) Groups of three C57BL/6 mice were immunized once with pEGFPL33A given i.m. (200 μg) or i.spl. (20 μg) or i.ln. (20 μg). Positive control mice received 500 pfu LCMV i.v. Ten days after immunization mice were challenged with 5 × 10⁴ pfu LCMV i.v., and 4 days later spleens were isolated and LCMV titers were determined. Symbols represent individual mice; one of two similar experiments is shown. (B) Groups of three C57BL/6 mice were immunized four times at 6-day intervals with pEFGPL33A DNA administered either i.m. (100 μg per immunization) or i.ln. (10 μg per immunization). Five days after the last immunization they were challenged with 5 × 10⁶ pfu Vacc-G2 i.p., and vaccinia titers in ovaries were assessed after a further 5 days. Symbols represent individual mice; one of two similar experiments is shown.

Figure 5

i.ln. immunization elicits protective antitumor immunity. Groups of six C57BL/6 mice were immunized three times at 6-day intervals with 10 μg of pEFGPL33A DNA or control pEGFP-N3 DNA. Five days after the last immunization small pieces of solid EL4–33 tumors were transplanted s.c. into both flanks, and tumor growth was measured every 3–4 days. Mean tumor volumes ± 1 SD are shown, and numbers in brackets indicate the ratio of the number of mice with tumors to the total number of mice in a group. One of two similar experiments is shown.