Vaccination with DNA encoding the immunodominant LACK parasite antigen confers protective immunity to mice infected with Leishmania major

Abstract

To determine whether DNA immunization could elicit protective immunity to Leishmania major in susceptible BALB/c mice, cDNA for the cloned Leishmania antigen LACK was inserted into a euykaryotic expression vector downstream to the cytomegalovirus promoter. Susceptible BALB/c mice were then vaccinated subcutaneously with LACK DNA and challenged with L. major promastigotes. We compared the protective efficacy of LACK DNA vaccination with that of recombinant LACK protein in the presence or absence of recombinant interleukin (rIL)-12 protein. Protection induced by LACK DNA was similar to that achieved by LACK protein and rIL-12, but superior to LACK protein without rIL-12. The immunity conferred by LACK DNA was durable insofar as mice challenged 5 wk after vaccination were still protected, and the infection was controlled for at least 20 wk after challenge. In addition, the ability of mice to control infection at sites distant to the site of vaccination suggests that systemic protection was achieved by LACK DNA vaccination. The control of disease progression and parasitic burden in mice vaccinated with LACK DNA was associated with enhancement of antigen-specific interferon-gamma (IFN-gamma) production. Moreover, both the enhancement of IFN-gamma production and the protective immune response induced by LACK DNA vaccination was IL-12 dependent. Unexpectedly, depletion of CD8(+) T cells at the time of vaccination or infection also abolished the protective response induced by LACK DNA vaccination, suggesting a role for CD8(+) T cells in DNA vaccine induced protection to L. major. Thus, DNA immunization may offer an attractive alternative vaccination strategy against intracellular pathogens, as compared with conventional vaccination with antigens combined with adjuvants.

Figure 1

LACK DNA vaccination provides similar protection as LACK protein plus rIL-12 to BALB/c mice infected with L. major. BALB/c mice (n = 12/group) were initally immunized and boosted 2 wk later with LACK DNA, control DNA (100 μg), or with LACK protein (50 μg) with or without rIL-12 (1 μg). 2 wk after the boost, mice were challenged in the hind footpad with 10⁵ live L. major metacyclic promastigotes. Weekly footpad measurements represent the average foot pad scores ± SEM.

Figure 2

Quantitation of viable parasite from draining lymph nodes. Single cell suspensions were made from draining lymph nodes harvested from infected mice at various time points after infection. The number of viable parasites was determined from the highest dilution at which promastigotes could be grown out 7 d of incubation. Data shown represents an average of three mice ± one SD at each time point.

Figure 3

Cytokine DNA and the amount of DNA used for vaccination influence disease outcome. (A) Mice (n = 12/ group) were immunized and boosted 2 wk later with varying doses of LACK DNA (10, 30, and 100 μg) in combination with varying amounts of control DNA to provide a total of 100 μg of DNA per vaccination. (B) In the same experiment, mice (n = 12/group) were immunized and boosted 2 wk later with LACK DNA (100 μg) plus 100 μg of control DNA, IL-4 DNA, or IL-12 DNA. 2 wk after the boost, mice were challenged in the hind footpad with 10⁵ live L. major metacyclic promastigotes.

Figure 3

Cytokine DNA and the amount of DNA used for vaccination influence disease outcome. (A) Mice (n = 12/ group) were immunized and boosted 2 wk later with varying doses of LACK DNA (10, 30, and 100 μg) in combination with varying amounts of control DNA to provide a total of 100 μg of DNA per vaccination. (B) In the same experiment, mice (n = 12/group) were immunized and boosted 2 wk later with LACK DNA (100 μg) plus 100 μg of control DNA, IL-4 DNA, or IL-12 DNA. 2 wk after the boost, mice were challenged in the hind footpad with 10⁵ live L. major metacyclic promastigotes.

Figure 4

LACK DNA vaccination induces a systemic and durable immune response to mice infected with L. major. (A) BALB/c mice (n = 6/ group) were initially immunized with LACK or control DNA (100 μg) and boosted 2 wk later. 5 wk after the boost, mice were challenged in the hind footpad with 10⁵ live L. major metacyclic promastigotes. (B) In a separate experiment, BALB/c mice (n = 6/group) were initially immunized and boosted 2 wk later with LACK or control DNA (100 μg) in the right hind footpad. 2 wk after the boost, mice were challenged with 10⁵ live L. major metacyclic promastigotes in the same or the opposite footpad to which they recieved LACK DNA vaccination.

Figure 4

LACK DNA vaccination induces a systemic and durable immune response to mice infected with L. major. (A) BALB/c mice (n = 6/ group) were initially immunized with LACK or control DNA (100 μg) and boosted 2 wk later. 5 wk after the boost, mice were challenged in the hind footpad with 10⁵ live L. major metacyclic promastigotes. (B) In a separate experiment, BALB/c mice (n = 6/group) were initially immunized and boosted 2 wk later with LACK or control DNA (100 μg) in the right hind footpad. 2 wk after the boost, mice were challenged with 10⁵ live L. major metacyclic promastigotes in the same or the opposite footpad to which they recieved LACK DNA vaccination.

Figure 5

In vitro production of IL-4 and IFN-γ from lymph node cells of vaccinated mice infected with L. major at 6 wk after infection. Individual mice (n = 3) were euthanized and the draining lymph nodes were harvested 6 wk after infection. Single cell preparations were plated in triplicate in 96-well microtiter plates at 3 × 10⁵ cells/200 μl in media alone or with LACK protein (10 μg/ml). 48 h later, supernatants were harvested and IFN-γ and IL-4 content were assayed by ELISA. Production of IFN-γ in media alone was <30 pg/ml. Production of IL-4 in media alone was usually 125 pg/ml or less. Data as shown represents the amount of IL-4 and IFN-γ averaged from three individual mice ± SEM. *P <0.005 in comparing IFN-γ produced from mice vaccinated with LACK DNA or LACK protein plus rIL-12 versus that produced from mice vaccinated with control DNA or LACK protein alone.

Figure 5

In vitro production of IL-4 and IFN-γ from lymph node cells of vaccinated mice infected with L. major at 6 wk after infection. Individual mice (n = 3) were euthanized and the draining lymph nodes were harvested 6 wk after infection. Single cell preparations were plated in triplicate in 96-well microtiter plates at 3 × 10⁵ cells/200 μl in media alone or with LACK protein (10 μg/ml). 48 h later, supernatants were harvested and IFN-γ and IL-4 content were assayed by ELISA. Production of IFN-γ in media alone was <30 pg/ml. Production of IL-4 in media alone was usually 125 pg/ml or less. Data as shown represents the amount of IL-4 and IFN-γ averaged from three individual mice ± SEM. *P <0.005 in comparing IFN-γ produced from mice vaccinated with LACK DNA or LACK protein plus rIL-12 versus that produced from mice vaccinated with control DNA or LACK protein alone.

Figure 6

LACK-specific production of IgG1 and IgG2A in vaccinated mice infected with L. major. Pooled sera (n = 7–12 mice per group) was collected 6 wk after infection from mice vaccinated with LACK DNA with or without IL-12 DNA, or LACK protein in the presence or absence of rIL-12. Sera was tested for LACK-specific antibody (A) IgG2a or (B) IgG1. Data shown is an average of duplicate absorbance (OD) values using serial fivefold dilutions.

Figure 6

LACK-specific production of IgG1 and IgG2A in vaccinated mice infected with L. major. Pooled sera (n = 7–12 mice per group) was collected 6 wk after infection from mice vaccinated with LACK DNA with or without IL-12 DNA, or LACK protein in the presence or absence of rIL-12. Sera was tested for LACK-specific antibody (A) IgG2a or (B) IgG1. Data shown is an average of duplicate absorbance (OD) values using serial fivefold dilutions.

Figure 7

Enhanced production of IFN-γ before infection is predictive of disease outcome. Pooled draining lymph nodes from mice (n = 5) were harvested 2 wk after the boost and before mice were infected with L. major. IL-4 and IFN-γ protein production were determined in a similar manner to that outlined in Fig. 5.

Figure 8

LACK DNA vaccination confers protection in an IL-12–dependent manner. (A) BALB/c mice immunized and boosted 2 wk later with LACK or control DNA (100 μg) were challenged with live 10⁵ L. major metacyclic promastigotes 2 wk after the boost. Some of the vaccinated mice (n = 10/group) were treated with 1 mg of anti–IL-12 intraperitoneally weekly from the time of vaccination to 4 wk after infection. (B) In the same experiment, draining lymph nodes from individual mice were harvested 6 wk after infection and production of IL-4 and IFN-γ were determined in a similar manner to that outlined in Fig. 5. *P <0.005 in comparing IFN-γ produced from mice vaccinated with LACK DNA versus that produced from mice vaccinated with LACK DNA plus anti– IL-12.

Figure 8

LACK DNA vaccination confers protection in an IL-12–dependent manner. (A) BALB/c mice immunized and boosted 2 wk later with LACK or control DNA (100 μg) were challenged with live 10⁵ L. major metacyclic promastigotes 2 wk after the boost. Some of the vaccinated mice (n = 10/group) were treated with 1 mg of anti–IL-12 intraperitoneally weekly from the time of vaccination to 4 wk after infection. (B) In the same experiment, draining lymph nodes from individual mice were harvested 6 wk after infection and production of IL-4 and IFN-γ were determined in a similar manner to that outlined in Fig. 5. *P <0.005 in comparing IFN-γ produced from mice vaccinated with LACK DNA versus that produced from mice vaccinated with LACK DNA plus anti– IL-12.

Figure 9

CD8⁺ T cells have an important role in mediating protection induced by LACK DNA vaccination. Mice (n = 10/group) receiving LACK or control DNA vaccination were treated with anti-CD8 antibody intraperitoneally starting at the time of vaccination (V) or at the time of infection (I) and then weekly until 4 wk after infection.