Conditions influencing the efficacy of vaccination with live organisms against Leishmania major infection

Abstract

Numerous experimental vaccines have been developed with the goal of generating long-term cell-mediated immunity to the obligate intracellular parasite Leishmania major, yet inoculation with live, wild-type L. major remains the only successful vaccine in humans. We examined the expression of immunity at the site of secondary, low-dose challenge in the ear dermis to determine the kinetics of parasite clearance and the early events associated with the protection conferred by vaccination with live L. major organisms in C57BL/6 mice. Particular attention was given to the route of vaccination. We observed that the rapidity, strength, and durability of the memory response following subcutaneous vaccination with live parasites in the footpad are even greater than previously appreciated. Antigen-specific gamma interferon (IFN-gamma)-producing T cells infiltrate the secondary site by 1.5 weeks, and viable parasites are cleared as early as 2.5 weeks following rechallenge, followed by a rapid drop in IFN-gamma(+) CD4(+) cell numbers in the site. In comparison, intradermal vaccination with live parasites in the ear generates immunity that is delayed in effector cell recruitment to the rechallenge site and in the clearance of parasites from the site. This compromised immunity was associated with a rapid recruitment of interleukin-10 (IL-10)-producing CD4(+) T cells to the rechallenge site. Treatment with anti-IL-10-receptor or anti-CD25 antibody enhanced early parasite clearance in ear-vaccinated mice, indicating that chronic infection in the skin generates a population of regulatory cells capable of influencing the level of resistance to reinfection. A delicate balance of effector and regulatory T cells may be required to optimize the potency and durability of vaccines against Leishmaniasis and other intracellular pathogens.

FIG. 1.

Protective immunity generated by FP vaccination with live parasites is characterized by the rapid clearance of parasites from a site of secondary challenge. Naive C57BL/6 mice (Na) or mice vaccinated 16 weeks prior in the RFP with 10⁴ L. major V1 metacyclic promastigotes (FP) were rechallenged, i.d., in both ears with 500 metacyclic promastigotes. Parasite loads were determined in the secondary ear dermis site (A) or DLN (B) at the indicated time points following rechallenge. Results shown represent parasite loads (per ear or DLN) for individual ears or DLNs. Three mice were employed per group per time point. ND, not done.

FIG. 2.

Protective immunity generated by FP vaccination with live parasites is characterized by the rapid recruitment of IFN-γ-producing CD4⁺ T cells to the site of secondary challenge. Naïve mice (Na) or mice vaccinated 16 weeks prior in the RFP with 10⁴ L. major metacyclic promastigotes (FP) were rechallenged in both ears with 500 metacyclic promastigotes. (A) At various time points following rechallenge, cells from the ear dermis were restimulated in vitro with L. major-infected dendritic cells and stained for surface markers and intracytoplasmic IFN-γ. (B) IFN-γ production by pooled cells from the DLN of the secondary site following in vitro restimulation with L. major-infected BMDDC as determined by ELISA.

FIG. 3.

Parasite clearance from a site of secondary challenge is delayed in mice vaccinated i.d. versus. s.c. Naïve mice (Na) or mice immunized 7 weeks prior in the FP or ear with 10⁴ live L. major metacyclic promastigotes were rechallenged in the contralateral ear (ear-vaccinated mice) or both ears (naïve and FP-vaccinated mice) with 10³ metacyclic promastigotes. At various intervals following intradermal rechallenge the number of parasites in ears (A) and DLN (B) of immune and naïve C57BL/6 mice was assessed. Results shown represent parasite loads for individual ears or DLNs. Data are for six mice per group per time point.

FIG. 4.

Recruitment of IFN-γ-producing CD4⁺ T cells to a site of secondary challenge is delayed in mice vaccinated i.d. versus s.c. Mice immunized 7 weeks prior in the FP or ear with 10⁴ live L. major metacyclic promastigotes were rechallenged in the contralateral ear or both ears with 10³ metacyclic promastigotes. At various time points following intradermal rechallenge, cells from the ear dermis were restimulated in vitro with L. major-infected BMDDC and stained for surface markers and intracytoplasmic IFN-γ.

FIG. 5.

Analysis of the rechallenge site in mice with chronic infection. Naïve mice (Na) or mice vaccinated 20 weeks prior with 10⁴ live L. major metacyclic promastigotes in the FP or ear were rechallenged in the contralateral ear or both ears with 10³ metacyclic promastigotes. Fourteen days following rechallenge, the secondary site was analyzed as follows: parasite load in the secondary ear dermis site (A); IFN-γ or IL-10 intracytoplasmic staining of CD4⁺ T cells isolated from the ear dermis following 24 h in vitro restimulation with BMDDC uninfected (DC) or infected with L. major (iDC) (B); IFN-γ production by dermal cells isolated from the secondary ear site measured by ELISA (C); and IL-10 production by dermal cells prepared as in panel C (D). Five mice per group were used. Results are representative of three repeat experiments. *, P = 0.0001 between iDC groups.

FIG. 6.

Treatment with anti-IL-10R or anti-CD25 antibodies leads to reduced parasite loads at the secondary site of infection in i.d. ear-vaccinated mice. C57BL/6 mice, vaccinated in the FP or ear 10 weeks prior with 10³ L. major metacyclic promastigotes were rechallenged in the contralateral ear with 10⁴ L. major promastigotes. Ear-vaccinated mice were treated with 0.5 mg of anti-CD25, anti-IL-10R, or isotype control (GL113) antibodies i.p., 3 days prior to, on the day of, and 3 days following rechallenge. Parasite load in the secondary ear site was determined 3 weeks following rechallenge. Each data point represents the secondary challenge site obtained from one mouse. Results are representative of three repeat experiments.

FIG. 7.

Analysis of the parasite load during the course of primary infection in ear- and FP-immunized mice. The number of parasites at the site of infection (A) and DLN (B) in C57BL/6 mice was determined at various time points following immunization with 10⁴ metacyclic L. major promastigotes in the ear or FP. Each data point represents an individual animal. *, P < 0.05; **, P < 0.005.

FIG. 8.

Analysis of L. major Ag-specific cytokine production by cells derived from the DLN of the site of chronic, primary infection. Naïve C57BL/6 mice were infected in the FP or the ear 16 weeks prior with 10⁴ L. major promastigotes. (A) IFN-γ production by DLN cells following in vitro restimulation with BMDDC infected (iDC) or uninfected (DC) with L. major as determined by ELISA. (B) IL-10 production by DLN cells as in panel A. *, P < 0.001 between iDC groups. The results are representative of two repeat experiments.