Testing of four Leishmania vaccine candidates in a mouse model of infection with Leishmania (Viannia) braziliensis, the main causative agent of cutaneous leishmaniasis in the New World

Abstract

We evaluated whether four recombinant antigens previously used for vaccination against experimental infection with Leishmania (Leishmania) major could also induce protective immunity against a challenge with Leishmania (Viannia) braziliensis, the species responsible for 90% of the 28,712 annual cases of cutaneous and mucocutaneous leishmaniasis recorded in Brazil during the year of 2004. Initially, we isolated the homolog genes encoding four L. (V.) braziliensis antigens: (i) homologue of receptor for activated C kinase, (ii) thiol-specific antioxidant, (iii) Leishmania elongation and initiation factor, and (iv) L. (L.) major stress-inducible protein 1. At the deduced amino acid level, all four open reading frames had a high degree of identity with the previously described genes of L. (L.) major being expressed on promastigotes and amastigotes of L. (V.) braziliensis. These genes were inserted into the vector pcDNA3 or expressed as bacterial recombinant proteins. After immunization with recombinant plasmids or proteins, BALB/c mice generated specific antibody or cell-mediated immune responses (gamma interferon production). After an intradermal challenge with L. (V.) braziliensis infective promastigotes, no significant reduction on the lesions was detected. We conclude that the protective immunity afforded by these four vaccine candidates against experimental cutaneous leishmaniasis caused by L. (L.) major could not be reproduced against a challenge with L. (V.) braziliensis. Although negative, we consider our results important since they suggest that studies aimed at the development of an effective vaccine against L. (V.) braziliensis, the main causative agent of cutaneous leishmaniasis in the New World, should be redirected toward distinct antigens or different vaccination strategies.

FIG. 1.

Comparison of the predicted amino acids 158 to 220 of the sti1 genes from L. (V.) braziliensis, L. (L.) major, or L. (L.) donovani. Alignments were obtained by the CLUSTAL W method. Residues that are different in the predicted amino acid sequences of L. (V.) braziliensis are boxed with a black background. Positions with substitutions between the L. (L.) major and L. (L.) donovani are boxed with gray background.

FIG. 2.

RNA expression of lack, tsa, leif, or Lbsti1 genes from L. (V.) braziliensis. Northern blot analysis was performed with lack (A), tsa (B), leif (C), lbsti (D), and α-tubulin (E) (12) ORFs as probes. Each lane contains 10 μg of total RNA of promastigotes of L. (L.) major (lane 1, positive control), promastigotes (lane 2) or amastigotes (lane 3) of L. (V.) braziliensis, or uninfected cultured macrophages (lane 4, negative control)/lane.

FIG. 3.

SDS-PAGE analyses of purified bacterial recombinant proteins from L. (V.) braziliensis. Proteins were submitted to SDS-PAGE under reducing conditions and stained with Coomassie blue. MW, molecular mass standard. The amount of each recombinant protein was approximately 600 ng per lane.

FIG. 4.

IIA recognition of promastigotes of L. (V.) braziliensis by immune sera from mice immunized with recombinant proteins. Parasites were incubated with a pool of sera derived from mice immunized with the respective recombinant protein and imaged under Nomarski differential interference contrast (A, C, E, G, I, and K) or fluorescence (B, D, F, H, J, and L). Magnification, ×1,000. Control sera was obtained from mice immunized with CFA or IFA alone. (M) Immunoblot of promastigote extract (30 μg of protein/lane) stained with a pool of sera from BALB/c mice immunized with the different recombinant proteins. Lanes: 1, LACK-immunized mice; 2, TSA-immunized mice; 3, LeIF-immunized mice; 4, LbSTI1-immunized mice; 5, mice immunized with all four antigens.

FIG. 5.

IIA recognition of amastigotes of L. (V.) braziliensis by immune sera from mice immunized with recombinant proteins. Mouse bone marrow macrophages infected with amastigotes of L. (V.) braziliensis were incubated with mouse antibodies specific to LACK (A to D), TSA (E to H), LeiF (I to L), or LbSTI1 (M to P). Additional slides were stained with negative (Q to T) or positive (U to X) control sera. All slides were costained with a pool of sera from humans infected with ML and with DAPI. Secondary antibodies to mouse IgG or human IgG were labeled with rhodamine (red fluorescence) or anti-FITC (green fluorescence), respectively. The images were captured by using Nomarski differential interference contrast (A, E, I, M, Q, and U) or fluorescence for rhodamine (B, F, J, N, R, and V) or FITC (C, G, R, O, S, and W). Fluorescence images for rhodamine, FITC, and DAPI (blue) were merged in panels D, H, L, P, T, and X. Magnification, ×1,000. Negative control sera were obtained from mice immunized with CFA or IFA alone.

FIG. 6.

Antibody immune response of mice immunized with eukaryotic expression plasmids or bacterial recombinant proteins expressing L. (V.) braziliensis antigens. (A) BALB/c mice (n = 6) were immunized with three doses of plasmids containing the indicated genes or pcDNA3 (control empty plasmid). Two weeks after the last immunization, blood samples were collected, and the sera were assayed by ELISA for the presence of antibodies to the respective recombinant proteins. Mice immunized with all plasmids were tested against the LbSTI1 recombinant protein only. (B) BALB/c mice (n = 7) were immunized with three doses of the indicated recombinant protein or adjuvant only (CpG ODN 1826 plus alum). Two weeks after the last immunization, blood samples were collected, and the sera were assayed by ELISA for the presence of antibodies to the respective recombinant proteins. Mice immunized with all proteins were tested in wells coated with all recombinant proteins. The results are presented as means ± the SD of reciprocal antibody dilutions (log₁₀) providing an optical density at 492 nm greater than 0.1.

FIG. 7.

IFN-γ secretion by spleen cells from BALB/c mice immunized with different plasmids or recombinant antigens of L. (V.) braziliensis upon in vitro restimulation with the respective recombinant proteins. BALB/c mice were immunized with three doses of plasmids containing the indicated genes (B) or pcDNA3 (A). Alternatively, BALB/c mice were immunized with three doses of the indicated recombinant proteins (D) or alum plus CpG ODN (C). Two weeks after the last immunizing dose, spleen cells of each individual mouse were stimulated in vitro with 10 μg of recombinant protein/ml. The concentration of IFN-γ was estimated in cell culture supernatants. The results are expressed as average of three mice ± the SD. This experiment was reproduced twice with identical results. Asterisks denote statistically significant differences from values in the supernatants of spleen cells cultured in the absence of the recombinant protein (P < 0.05, one-way analysis of variance).

FIG. 8.

Lesion size caused by infective promastigotes of L. (V.) braziliensis in BALB/c mice immunized with eukaryotic expression plasmids, followed by bacterial recombinant proteins expressing Leishmania antigens. BALB/c mice were immunized twice intramuscularly with 100 μg per dose of the indicated plasmids or pcDNA3 (control). These animals were subsequently immunized intraperitoneally once (A) or twice (B) with the respective recombinant proteins (25 μg/dose) admixed with CpG ODN 1826 and alum or adjuvant alone (control). Two weeks after the last immunizing dose, mice were challenged i.d. in both ears with 10⁵ promastigotes of L. (V.) braziliensis (strain MHOM/BR/01/BA788). The results are expressed as the average lesion size of three mice ± the SD. No statistically significant difference was observed when we compared the distinct mouse groups (P > 0.05, one-way analysis of variance).