Toward a defined anti-Leishmania vaccine targeting vector antigens: characterization of a protective salivary protein

Abstract

Leishmania parasites are transmitted to their vertebrate hosts by infected phlebotomine sand fly bites. Sand fly saliva is known to enhance Leishmania infection, while immunity to the saliva protects against infection as determined by coinoculation of parasites with vector salivary gland homogenates (SGHs) or by infected sand fly bites (Kamhawi, S., Y. Belkaid, G. Modi, E. Rowton, and D. Sacks. 2000. Science. 290:1351-1354). We have now characterized nine salivary proteins of Phlebotomus papatasi, the vector of Leishmania major. One of these salivary proteins, extracted from SDS gels and having an apparent mol wt of 15 kD, was able to protect vaccinated mice challenged with parasites plus SGH. A DNA vaccine containing the cDNA for the predominant 15-kD protein (named SP15) provided this same protection. Protection lasted at least 3 mo after immunization. The vaccine produced both intense humoral and delayed-type hypersensitivity (DTH) reactions. B cell-deficient mice immunized with the SP15 plasmid vaccine successfully controlled Leishmania infection when injected with Leishmania plus SGH. These results indicate that DTH response against saliva provides most or all of the protective effects of this vaccine and that salivary gland proteins or their cDNAs are viable vaccine targets against leishmaniasis.

Figure 1

SDS-PAGE and Western blots of SGH of P. papatasi. (A) Coomassie blue–stained PVDF membrane after gel transfer of 40 homogenized pairs of glands. The numbers represent the position of the mol wt markers. (B) Western blots of the gels with mouse sera obtained from mice immunized by intradermal inoculation of SGH (needle inoculation) or the bite of uninfected sand flies (sand fly bite) with their preimmunization (PI) controls.

Figure 2

SDS-PAGE of 20 homogenized pairs of salivary glands from P. papatasi. Left side of figure, indication of the NH₂-terminal sequence found for each band by Edman degradation; right side of figure, clone identification name. Numbers indicate the position of the mol wt marker in the same gel.

Figure 3

Effect of mouse immunization with fractions of SGH separated by SDS-PAGE. Effect on (A) the lesion size and (B) the number of parasites 4.5 wk postchallenge after L. major infection. Naive mice were inoculated intradermally with 500 L. major promastigotes alone (○) or in the presence of 0.5 pairs of SGH (•). Mice previously vaccinated on the right ear (2-wk intervals) with acrylamide alone, E (▪) or mol wt range from 200 to 40 kD, A (♦); 39 to 20 kD, B (⋄); or 19 to 3 kD, C (▴) were challenged in the left ear 2 wk after the last immunization with 500 L. major promastigote in the presence of 0.5 pairs of SGH. The above symbols and bars represent the mean induration in mm ± SE (five mice per group) or the mean number of parasite per ear ± SE (four mice per group). (*) indicate significance at P < 0.05 when treatment curve (C) was compared with the acrylamide control (E).

Figure 4

Western blots of SGH of P. papatasi reacted with sera from mice immunized with polyacrylamide fractions separating the SGH by SDS-PAGE. Divisions were a high (A), medium (B), and low (C) mol wt region, as detailed in the legend to Fig. 3, or from control mice immunized with polyacrylamide (E), as shown.

Figure 5

Effect of mouse immunization with band SP15 from SDS-PAGE gels separating the SGH of P. papatasi on the lesion size (A) and the number of parasites 9 wk postchallenge (B) after L. major infection. Naive mice were inoculated intradermally with 500 L. major promastigotes alone (○) or in the presence of 0.5 pairs of SGH (•). Mice previously vaccinated on the right ear (2-wk intervals) with acrylamide alone, E (□) or band SP15 (▪) were challenged in the left ear 2 wk after the last immunization with 500 L. major promastigotes in the presence of 0.5 pairs of SGH. The above symbols and bars represent mean induration in mm ± SE (five mice per group) or mean number of parasites per ear ± SE (five mice per group). (*) indicate significance at P < 0.05 when the treatment curve was compared with the acrylamide control.

Figure 6

Mouse humoral and cellular immunities are generated by injection of the VR1020 plasmid containing the SP15 sequence (SP15-Pl). (A) Western blots of salivary homogenates of P. papatasi reacted against sera of mice immunized twice in the right ear (2-wk interval) with 5 μg of the SP15-Pl or control VR1020 plasmid (CTL-Pl). (B) DTH reaction induced by sand fly bites on mice vaccinated with SP15-Pl. The left ears of naive mice, mice immunized with SP15-Pl, or control CTL-Pl mice were exposed to the bite of 10 sand flies. 24 h later, the mice were killed, and the inflammatory cells (neutrophils, eosinophils, macrophages, dendritic cells, CD4⁺, and CD8⁺ T cells) present in the dermis were analyzed. The symbols and bars represent the mean number of cellular subsets per ear ± SE; seven mice per group.

Figure 7

Effect of mouse immunization with SP15-Pl on the lesion size caused by L. major infection. Naive mice were inoculated intradermally with 500 L. major promastigotes alone (○) or in the presence of 0.5 pairs of SGH (•). Mice previously vaccinated on the right ear (2-wk intervals) with SP15-Pl (□, ▪) or CTL-Pl (▵, ▴) were challenged in the left ear 2 wk after the last immunization with 500 L. major promastigotes in the presence (▪, ▴) or not (□, ▵) of 0.5 pairs of SGH. The symbols and bars represent the mean induration in mm ± 1 SE; five mice per group. (*) indicate significance at P < 0.05 when the treatment curve was compared with the control (CTL-Pl + SGH).

Figure 9

Humoral response and DTH reaction on B^−/− and wt mice after vaccination with SP15-Pl. Western blots showing antibody reactivity of wt but not B^−/− mice against P. papatasi salivary homogenates (A). Measurements indicated the millimetric difference between the ear challenged with SGH and the noninoculated ear, on B^−/− and wt C57BL/10 mice vaccinated with SP15-Pl (B). Mice were immunized twice in the right ear (2-wk intervals) or not with 5 μg of SP15-Pl or CTL-Pl and challenged in the left ear 2 wk after the last immunization with 500 L. major promastigotes in the presence of 0.5 pairs of SGH. 24 h after inoculation, the ear thickness was measured and the difference between the ear thickness before challenge and 24 h after challenge was computed. Symbols and bars represent mean thickness in mm ± SE; five mice per group.

Figure 9

Humoral response and DTH reaction on B^−/− and wt mice after vaccination with SP15-Pl. Western blots showing antibody reactivity of wt but not B^−/− mice against P. papatasi salivary homogenates (A). Measurements indicated the millimetric difference between the ear challenged with SGH and the noninoculated ear, on B^−/− and wt C57BL/10 mice vaccinated with SP15-Pl (B). Mice were immunized twice in the right ear (2-wk intervals) or not with 5 μg of SP15-Pl or CTL-Pl and challenged in the left ear 2 wk after the last immunization with 500 L. major promastigotes in the presence of 0.5 pairs of SGH. 24 h after inoculation, the ear thickness was measured and the difference between the ear thickness before challenge and 24 h after challenge was computed. Symbols and bars represent mean thickness in mm ± SE; five mice per group.

Figure 8

Long-term effect of mouse immunization with SP15-Pl on L. major infection. (A) Effect on lesion size. Naive mice were inoculated intradermally with 500 L. major promastigotes alone (○) or in the presence of 0.5 pairs of SGH (•). Mice previously vaccinated on the right ear (2-wk intervals) with SP15-Pl (□, ▪) or CTL-Pl (▵, ▴) were challenged in the left ear 12 wk after the last immunization with 500 L. major promastigotes in the presence (▪, ▴) or not (□, ▵) of 0.5 pairs of SGH. (B) Effect on parasite number at 6.5 wk postchallenge. The symbols and bars represent the mean induration in mm ± 1 SE; five mice per group. (*) indicate significance at P < 0.05 when the treatment curve was compared with the control (CTL-Pl + SGH).

Figure 10

Role of DTH in mouse immunization with SP15-Pl on subsequent L. major infection. B^−/− mice (▵, ▴) and their controls (C57BL/10, wt; □, ▪), were immunized twice in the right ear (2-wk interval) with the VR1020 plasmid with (filled symbols) or without (open symbols), the SP15 sequence and challenged 2 wk later in the left ear with L. major promastigotes in combination with 0.5 pairs of homogenized P. papatasi salivary glands. (A) Lesion size progression. (B) Parasite numbers recovered from the lesion at 5.5 wk. Each number and bar represents the average ± SE of five mice. (*) indicates significance at P < 0.05 when the number of parasites on the SP15-Pl group was compared with the controls of the same mouse group.