Balb/C BMDCs were cultured with titrating concentrations of either TLR4 agonists (MPL or GLA) or TLR 9 agonist (CpG) or a combination of both. Superantants were collected after 24 hrs and assayed for IL-12p70. Data is representative of 3 independent experiments.

1. Naïve (a) or Infected (b&c) Balb/C mice were immunized weekly and harvested 1 week post third immunization. Individual spleens and pooled draining LNs were stimulated ex vivo for 12 hours and analyzed by ICS for activated CD44^hiIFN-γ++CD4 T cells. Percent CD4 T cells positive for CD44 and IFN-γ are plotted. *p values ≤ 0.04, ** p values ≤ 0.06, *** p values ≤ 0.001. Data is representative of 2 independent experiments.
2. Naïve (a) or Infected (b) Balb/C mice were immunized weekly and harvested 1 week post the fifth immunization. Spleens were stimulated ex vivo for 12 hours and analyzed by ICS for activated CD44^hiIFN-γ++CD4 T cells. Percent CD4 T cells positive for CD44 and IFN-γ are plotted. * p values <0.005; **p values <0.001;***p value < 0.05; Data is representative of 3 independent experiments.

1. Naïve Balb/C mice were immunized weekly and spleens harvested 1 week post-third (a) or -fifth immunization (b) stimulated ex-vivo for 72 hours. Supernatants were collected and assayed for cytokines, IFN-γ and IL-13. Vaccine draining inguinal LNs (pooled) were also harvested and assayed similarly at either 1 week post- third (c) or -fifth immunization (d). Data is representative of 2 independent experiments.
2. Infected Balb/C mice were immunized weekly and draining popliteal LNs (pooled) were harvested 1 week post- third (a) or -fifth immunization (b) stimulated ex-vivo for 72 hours and supernatants were assayed for cytokines, IFN-γ and IL-13. Data is representative of 2 independent experiments.

Infected Balb/C mice were immunized weekly and parasite-draining popliteal LNs (a) and footpads (b) were harvested week post- third immunization for RNA isolation. Samples were assayed for IL-12, IFN-γ, IL-10 and RANTES (CCL5) by RT-PCR. *p value < 0.05; Data is representative of 2 independent experiments.

Balb/c mice were infected in the hind-footpads with 1–2*10⁵ metacyclic promastigotes of L. major. One week post-infection, mice were immunized, weekly with either control saline or various vaccine formulations and foot pads measured every week prior to vaccination (a). Footpads were harvested at the end of the experiment by limiting dilution to estimate parasite burden (b). Footpad lesion sizes of saline treated (top) vs. vaccine-containing adjuvant combo treated (bottom) are shown (c).* p values <0.0001
Data is representative of 4 independent experiments.

1. Gating strategy to identify Multi-functional T cells versus Terminal effectors: Using Multi-parameter flow cytometry, CD44^hiIFN-γ++CD4 T cells were further analyzed for IL-2 and TNF-α production.
2. Naïve mice were immunized weekly with L110f in combination with different adjuvant(s) and harvested 1 week post third immunization and splenocytes stimulated ex vivo for 12 hours and analyzed by ICS (a). Infected mice were immunized weekly and harvested 1 week post third immunization and splenocytes stimulated ex vivo for 12 hours and analyzed by ICS (b). Cells from pooled inguinal LNs (c) and pooled popliteal LNs (d) of infected mice were also stimulated ex vivo for 12 hours and analyzed by ICS. Percent CD44^hiIFN-γ++CD4 T cells positive for IFN-γ and/or IL-2 and/or TNF-α are plotted for all organs. n.d.-none detected. Data is representative of 2 independent experiments.
3. Naïve (a) or Infected (b) Balb/C mice were immunized weekly with L110f in combination with different adjuvant(s) and harvested 1 week post fifth immunization. Splenocytes were stimulated ex vivo for 12 hours and analyzed by ICS for activated CD44hiIFN-γ++CD4 T cells. Percent CD44^hiIFN-γ++CD4 T cells positive for IFN-γ and/or IL-2 and/or TNF-α are plotted. n.d.-none detected. Data is representative of 3 independent experiments.
4. Flowdata from Week 4 were reanalyzed for to calculate ratios of terminal effectors (Only IFN-γ++) to bifunctional T cells (IFN-γ +TNF-α+; closed bars) or multifunctional T cells (IFN-γ+TNF-α+IL-2+; open bars) in the absence (a) or presence of infection (c). reanalyzed for to calculate ratios of terminal effectors (Only IFN-γ++) to bifunctional T cells (IFN-γ+TNF-α+;closed bars or multifunctional T cells (IFN-γ+TNF-α+IL-2+; open bars) in the absence (b) or presence of infection (d). * p value < 0.01; ** p value ≤ 0.05; ***p value < 0.1; n.d.-none detected. Data is representative of 2 independent experiments.

Balb/c BMDCs were stimulated with titrating concentrations of MPL ± CpG (μg/ml) for 24 hrs. Supernatants were assayed for a series of Th1/Th2 cytokines (a) and chemokines (a&b) using a Multiplex platform. Data is representative of 2 independent experiments.