Subunit vaccination of mice against new world cutaneous leishmaniasis: comparison of three proteins expressed in amastigotes and six adjuvants

Abstract

A mixture of well-defined recombinant antigens together with an adjuvant that preferentially stimulates specific gamma interferon (IFN-gamma)-secreting helper type 1 CD4(+) T cells (Th1 cells) presents a rational option for a vaccine against leishmaniasis. The potential of this approach was investigated in murine infections with Leishmania mexicana, which are characterized by the absence of a parasite-specific Th1 response and uncontrolled parasite proliferation. A mixture of three antigens (glycoprotein 63, cysteine proteinases, and a membrane-bound acid phosphatase), which are all expressed in amastigotes, the mammalian stage of the parasite, were used for the immunization of C57BL/6 mice in combination with six adjuvants (interleukin 12 [IL-12], Detox, 4'-monophosphoryl lipid A, QS-21, Mycobacterium bovis BCG, and Corynebacterium parvum). All six vaccine formulations containing the mixture of recombinant antigens were protective against challenge infections with promastigotes, the insect stage of the parasite, in that mice controlled and healed infections but developed transient and, in certain cases, accentuated disease. The most effective adjuvants were IL-12 followed by Detox. Further studies using these two adjuvants showed that a similar protective effect was observed with a mixture of the corresponding native proteins, and mice which had controlled the infection showed a preponderance of IFN-gamma-secreting CD4(+) T cells in the lymph nodes draining the lesion. Using the recombinant proteins individually, it is shown that the relatively abundant cysteine proteinases and glycoprotein 63, but not the acid phosphatase, are able to elicit a protective response. The results are discussed in comparison to previous studies with subunit vaccines and with respect to cell biological aspects of antigen presentation in Leishmania-infected macrophages.

FIG. 1

Antigen-specific T cells are induced in mice by immunization with a mixture of recombinant proteins and different adjuvants. The mice were immunized with formulations containing all three recombinant proteins and the adjuvants as indicated in the figure (see Materials and Methods). Single cell suspensions of spleens (A) and lymph nodes (B) of vaccinated mice were restimulated in vitro in the presence of rMBAP, rCP5, rGP63 or a freeze-thawed lysate of L. mexicana as indicated. Cell proliferation was assessed by measuring [³H]thymidine incorporation. Antigen-specific cell proliferation is expressed by stimulation indices calculated from the incorporated radioactivity in the presence of antigen divided by the activity incorporated in cells cultured in medium only (background 5 × 10³ to 2.6 × 10⁴ cpm/culture well). Data represent the arithmetic mean of triplicate determinations.

FIG. 2

Efficacy of different vaccine formulations containing rMBAP, rCP5, and rGP63. C57BL/6 mice were injected twice at a weekly interval into the lower back with 200 μl of vaccine containing 2.5 μg of each protein mixed with the indicated adjuvants. One week after the boosting injection, 4 × 10⁵ L. mexicana promastigotes were injected into the hind left footpad. Footpad swelling was measured every second week, and the data represent the mean increase in thickness compared to the uninfected foot of five mice per group. One animal in the groups treated only with QS-21, Detox, and C. parvum showed a tendency to heal at the end of the experiment. However, the averaged values include all mice per group. The plots are arranged in two graphs: lesion development in groups immunized with QS-21, Detox, or C. parvum (A) and in groups immunized with BCG, MPL, or IL-12 (B). PBS controls are shown in both graphs.

FIG. 3

Protective efficacy of vaccines containing native antigens and IL-12 or Detox as adjuvants. C57BL/6 mice (five per group) were vaccinated with two doses of 2.5 μg each of CP, GP63, and MBAP mixed with IL-12 or Detox as described in the text. Immunized animals were infected with 4 × 10⁵ L. mexicana promastigotes. Lesion development was monitored as outlined in Fig. 2. In the group treated with antigen (Ag) and Detox, two of five animals still had active lesions at the end of the experiment, while one of five mice showed a tendency to heal in the groups treated only with IL-12 or Detox or PBS-antigen (PBS-Ag). Error bars are shown for one group only for better clarity and correspond to the mean ± the standard deviation (SD), including all mice, and are representative for all groups.

FIG. 4

Protective efficacy of vaccines in mice challenged with different doses of L. mexicana. C57BL/6 mice were vaccinated with a mixture of the three recombinant antigens and IL-12 or Detox as adjuvants as described in Fig. 2 and challenged with 4 × 10⁵ (A) or 2 × 10⁶ (B) L. mexicana promastigotes. Lesions were scored as in Fig. 2, and data represent the average values of five mice per group. Error bars are shown for representative groups and correspond to the mean ± the SD.

FIG. 5

Comparison of IFN-γ secretion by CD4⁺ T cells of protected and nonprotected C57BL/6 mice. CD4⁺ T cells were prepared from lymph nodes draining the site of infection of cured mice vaccinated with IL-12 and rMBAP, rCP, and rGP63 (see Fig. 2) or of PBS mock-treated mice with progressive disease. For control purposes, CD4⁺ T cells were also enriched from corresponding lymph nodes of naive, noninfected animals. The enriched cell populations were stimulated with a freeze-thawed lysate of L. mexicana promastigotes presented by syngeneic splenocytes in vitro. Antigen-specific IFN-γ production was determined in the supernatants and compared to the production of this cytokine in cultures treated with concanavalin A or medium alone. Values represent the mean of duplicate cultures of CD4⁺ cells from individual mice (three animals of each infected group and two naive controls). The lymphokine content varied by less than 10% between duplicates. Antigen-specific IFN-γ production by CD4⁺ cells from vaccinated mice was significantly higher than that by cells from mock-treated mice (P ≤ 0.05; in a Wilcox ranking test).

FIG. 6

Efficacy of rMBAP, rCP5, or rGP63 in combination with IL-12 as single-component subunit vaccines. C57BL/6 mice (five per group) were vaccinated with two doses of 5 μg of the individual proteins mixed with IL-12. Control mice were mock immunized with IL-12 in PBS or with PBS alone. Mice were challenged 1 week later with 4 × 10⁵ L. mexicana promastigotes. Lesion development was monitored as outlined in Fig. 2. Error bars are shown for the two protected groups and correspond to the mean ± the SD.