Leishmania major strains isolated from distinct endemic areas show diverse cytokine mRNA expression levels in C57BL/6 mice: Toward selecting an ideal strain for the vaccine studies.

Leishmania major, the causative agent of zoonotic cutaneous leishmaniasis shows heterogeneity and diverse clinical manifestations in different areas of infection and experimental models. Such polymorphism may cause difficulties in selection of reliable strains for development of prophylaxes. Hence, the aim of this study was to identify an ideal strain of L. major, capable of inducing protective and long-lasting Th1 responses in an animal model that mimics the human response to L. major infection. The isolates were from patients residing in 4 endemic areas of L. major in Iran, namely Damghan (north), Kashan (center), Dehloran (west) and Shiraz (south) which their heterogeneity had been previously confirmed in BALB/c mice. In this study, the same isolates as well as the Iranian reference strain of L. major were inoculated to C57BL/6 mice to evaluate their pathogenicity and changes in expression of key cytokine genes from lymph nodes of the mice in different time points, in order to evaluate their ability to control leishmaniasis by development of Th1 responses. Our results showed the lowest and highest parasite burden in lymph nodes of mice infected with all strains at weeks 3 and 8 post-infection, respectively. However, the Damghan strain (DA39) showed comparatively lower number of viable parasite than other strains at week 8 post-infection. Furthermore, DA39 showed higher expression of Ifng and Il12 mRNA at week 8 post-infection while the ratio of its Ifng/Il4 mRNA expressions was higher than other strains. In conclusion, DA39 among the studied strains appears to induce strong and lasting Th1 cytokine gene expressions with minimum virulence, making it a suitable candidate strain for vaccine studies in leishmaniasis.

Evaluation of Cellular Immune Responses in Dogs Immunized with Alum-Precipitated Autoclaved Leishmania major along with BCG and Imiquimod.

BACKGROUND: We aimed to investigate the potential effects of BCG and imiquimod on improvement of current experimental L. major vaccine against dogs in an endemic area of Zoonotic visceral leishmaniasis (ZVL) in Iran. METHODS: During 2012 till 2014, seven mixed-breed shepherd dogs with no anti-Leishmania antibodies and no response to Leishmanin reagent were immunized with 2 doses of alum-precipitated autoclaved L. major (Alum-AML) while BCG and imiquimod (for skin pre-treatment) were used as adjuvants. The productions of a few characteristic cytokines of T-helper immune responses and the development of delayed-type hypersensitivity (DTH) of the immunized animals were then evaluated, up to 300 days. Blood samples were collected at 0, 30, 80 and 300 d post-vaccination and the concentrations of IFN-γ, IL10, IL-12 and TGF-ß cytokines secreted from PBMCs at these time-points were quantified by ELISA. DTH was evaluated by Leishmanin skin test (LST). RESULTS: Although a similar LST conversion was observed at all time-points, the cytokine measurement results indicated significantly higher levels of IFN-γ at day 80 and elevated levels of IL-10 at days 80 and 300, post-vaccination. Moreover, a significantly higher IFN-γ/IL-10 ratio was observed at day 30 post-vaccination compared to the other time-points. CONCLUSION: Although a Th1-like response could be observed at day 30 post-vaccination, the development of cytokine profiles was inclined toward mixed Th1 and Th2 responses at days 80 and 300 post-vaccination. This situation may indicate the requirement of an additional boosting by this Alum-AML formula, in order to induce long-lasting protection against ZVL.

Naloxone Diminishes the Virulence and Modifies the Cellular Immune Responses of BALB/c Mice Infected with Leishmania major.

PURPOSE: Leishmania major-infected BALB/c mice display strong susceptibility to the infection due to the induction of Th2 response. The aim of this study was to assess the effects of naloxone on virulence of L. major in BALB/c mice and the ensued cellular immune response. METHODS: The effects of injection of a single dose of naloxone in the footpad of L. major-infected BALB/c mice were investigated by evaluating the lesion sizes, the parasite burden, cell proliferation, secreted cytokines (IFN-γ, IL-4, IL-10 and IL-12) and their genes expressions due to naloxone treatment while the untreated mice were used as a control. RESULTS: Significantly lower lesion sizes and less parasite burden were measured in the treated mice. Significantly decreased productions of IFN-γ, IL-12, IL-4, and IL-10 were also observed in the treated mice at week 4 post-infection while the production IL-10 remained significantly hindered till 8 weeks post-infection. CONCLUSION: Our data indicated that although the treatment of L. major-infected BALB/c mice with a single dose of naloxone was unable to improve the cellular immune response, it led to lower virulence, confirmed by significantly reduced lesions and parasite load.

FOXP3 expression and frequency of regulatory T cells in healed individuals from Leishmania major infection and the asymptomatic cases.

Two groups of residents in an endemic area of Leishmania major infection in Iran with positive leishmanin skin tests who were either asymptomatic or had healed cutaneous leishmaniasis lesions were compared with respect to their T helper responses. The percentages of regulatory T cells (Treg; CD4(+)CD25(high) FoxP3(+)) from the peripheral blood and CD4(+) T cells producing intracellular cytokines (IL-4, IL-10, IL-17 and IFN-γ) from the stimulated PBMCs were evaluated by flow cytometry and the expressions of RORC and FOXP3 genes were quantified by real-time RT-PCR. T responder (CD4(+)CD25(-)) and Treg-enriched (CD4(+)CD25(+)) cells were isolated magnetically and the suppressive capacity of the latter and the cytokines (IFN-γ, TGF-ß and IL-10) secreted from them were evaluated by in vitro assays. The results showed that the frequency of Treg in the studied groups were similar and Treg from both groups exhibited high yet similar suppressive capacities while significantly higher levels of FOXP3 expression was observed in the asymptomatic group. Taken together, similar frequency and suppressiveness of Treg combined with high ratios of IFN-γ/IL-10 producing CD4(+) T cells were common in both groups; however the members of the asymptomatic group appeared to require higher expression of FOXP3 to maintain their immunity to re-infection.

Ferroportin-encapsulated nanoparticles reduce infection and improve immunity in mice infected with Leishmania major.

Inoculation of inbred mice by Leishmania major results in two different patterns. C57BL/6 mice display resistance against L. major but BALB/c mice show susceptibility to L. major with visceral infection, anemia and death. In this study, the effects of treatment of L. major-infected BALB/c mice with a ferroportin (Fpn)-encoding construct via nanoparticles were evaluated. A fragment encoding Fpn, a major regulator of iron homeostasis, was amplified and sub-cloned to a GFP expression vector to express Fpn-EGFP protein. This construct was incorporated in nanoparticles of alginate/chitosan polymers and orally administered to L. major-infected BALB/c mice. Blood hematocrit and iron, footpad size, parasite load and concentration of IFNG, IL4 and IL10 by ELISA were measured in the treated and untreated mice. The results indicated that the treated mice had significantly higher hematocrit and iron levels while exhibited significantly lower footpad size and parasite load measurements. Moreover, lower levels of IL4 and IL10 and higher ratios of IFNG/IL4 or IFNG/IL10 were shown in the treated, compared to the untreated mice. In conclusion, treating BALB/c mice infected with L. major with encapsulated Fpn-encoding construct in alginate/chitosan nanoparticles were shown to reduce the infection and improve anemia and immunity in the animal model of leishmaniasis.

Genotypically distinct strains of Leishmania major display diverse clinical and immunological patterns in BALB/c mice.

Infection with Leishmania major species is endemic in many provinces of Iran. Isolates from four endemic areas located in north (Damghan), center (Kashan), west (Dehloran), and south (Shiraz) of country which showed major distinctive polymorphism by RAPD-PCR method were evaluated. Isolates were inoculated to different groups of BALB/c mice and their clinical and immunological status was compared. Lesion size, parasite burden and T cell phenotype in lymph node (LN), and cytokine secretion in the culture of LN mononuclear cells were determined. The results showed the lowest and highest lesion sizes in mice infected by Shiraz strain (3.02+/-0.52 mm) and Kashan strain (5.20+/-0.45), respectively, 8 weeks after inoculation. No significant difference was observed between other strains. The parasite burden was significantly lower in lymph node of mice infected with strain of Damghan (1.51 x 10(7)) than Kashan (3.60 x 10(9)) and Shiraz (7.08 x 10(9)) strains, 8 weeks post-infection. However, Dehloran strain showed intermediate load of viable parasites (1.51 x 10(9)) in LN, 8 weeks post-infection. High ratios of IFN-gamma/IL-4 were shown in mice inoculated by strain of Dehloran (3.17) and Damghan (2.66), but not in mice infected by other strains, 8 weeks post-infection. The highest and lowest ratios of CD4(+)/CD8(+) T cells were found in LN cells of mice infected with Kashan (1.82) and Dehloran (1.00) strains, respectively. Results indicate that the lowest and intermediate loads of parasites induced by Damghan and Dehloran strains along with higher ratio of IFN-gamma/IL-4 produced by both strains in LN of inoculated mice suggest that these strains have the capacity to shift the immune responses to a predominant Th1 response after 8 weeks infection in BALB/c mice and might be the ideal strains for vaccine studies and development of candidate vaccine against leishmaniasis.

Exposure to Leishmania major modulates the proportion of CD4+ T cells without affecting cellular immune responses.

The immune responses of individuals exposed to Leishmania major were evaluated and compared with those of non-exposed volunteers. Forty-one patients with active lesion(s), 43 healed individuals, 15 vaccinees 1 month or 1 year post vaccination, and 15 non-exposed volunteers were studied. Leishmanin skin test (LST) response, proliferative response of lymphocyte (PRL) to L. major antigen, IFN-gamma and IL-4 production, and percentage of L. major-specific CD4+, CD8+ and CD16+/CD56+ cells in peripheral blood mononuclear cells were assessed. Data showed positive LST (>5 mm) in 92% of patients, 98% of healed, and 80% or 43% of vaccinees 1 month and 1 year post vaccination, respectively. Positive PRL (SI>2.5) was displayed in 90%, 84%, 46% and 7% of patients, healed, vaccinated (post 1 year) and non-exposed donors, respectively. The mean +/-S.E. of IFN-gamma was 924 +/- 149, 1,278 +/- 185, 470 +/- 282 or 258 +/- 82 pg/ml in patients, healed cases and vaccinees after 1 month or 1 year, respectively. Positive IFN-gamma responders (>300 pg/ml) were shown in 72% of patients, 81% of healed cases, 31% or 39% of vaccinees and 0% of non-exposed donors. A reduced percentage of CD4+ T-cells and an increased percentage of NK cells were found in exposed individuals compared to non-exposed donors. The data indicated that exposure to L. major modulates the proportion of CD4+ T cells and increases NK cells percentage. However, the cellular immune responses including induction of LST, and IFN-gamma production are increased in exposed individuals.

The role of BCG in human immune responses induced by multiple injections of autoclaved Leishmania major as a candidate vaccine against leishmaniasis.

To determine if BCG was required in booster injections for autoclaved Leishmania major (ALM) vaccine, 75 volunteers with no response to leishmanin were injected double-blind and randomly with either ALM+BCG or BCG alone for the first injection and boosted either with ALM+BCG, ALM or BCG alone for the second and third. Addition of BCG to the boosters significantly increased the frequency and the magnitude of leishmanin skin tests (LSTs); however, there was no difference in proliferative and IFN-gamma responses (a month and a year later). Three injections of BCG produced no observable adverse reaction; hence BCG could be used in booster injections to increase the protective potential of this candidate vaccine.