TLR1/2 activation during heterologous prime-boost vaccination (DNA-MVA) enhances CD8+ T Cell responses providing protection against Leishmania (Viannia)

Abstract

Background: Leishmania (Viannia) parasites present particular challenges, as human and murine immune responses to infection are distinct from other Leishmania species, indicating a unique interaction with the host. Further, vaccination studies utilizing small animal models indicate that modalities and antigens that prevent infection by other Leishmania species are generally not protective.

Methodology: Using a newly developed mouse model of chronic L. (Viannia) panamensis infection and the heterologous DNA prime - modified vaccinia virus Ankara (MVA) boost vaccination modality, we examined whether the conserved vaccine candidate antigen tryparedoxin peroxidase (TRYP) could provide protection against infection/disease.

Results: Heterologous prime - boost (DNA/MVA) vaccination utilizing TRYP antigen can provide protection against disease caused by L. (V.) panamensis. However, protection is dependent on modulating the innate immune response using the TLR1/2 agonist Pam3CSK4 during DNA priming. Prime-boost vaccination using DNA alone fails to protect. Prior to infection protectively vaccinated mice exhibit augmented CD4 and CD8 IFNγ and memory responses as well as decreased IL-10 and IL-13 responses. IL-13 and IL-10 have been shown to be independently critical for disease in this model. CD8 T cells have an essential role in mediating host defense, as CD8 depletion reversed protection in the vaccinated mice; vaccinated mice depleted of CD4 T cells remained protected. Hence, vaccine-induced protection is dependent upon TLR1/2 activation instructing the generation of antigen specific CD8 cells and restricting IL-13 and IL-10 responses.

Conclusions: Given the general effectiveness of prime-boost vaccination, the recalcitrance of Leishmania (Viannia) to vaccine approaches effective against other species of Leishmania is again evident. However, prime-boost vaccination modality can with modulation induce protective responses, indicating that the delivery system is critical. Moreover, these results suggest that CD8 T cells should be targeted for the development of a vaccine against infection caused by Leishmania (Viannia) parasites. Further, TLR1/2 modulation may be useful in vaccines where CD8 T cell responses are critical.

Figure 1. Up regulation of Th1 cytokines and down regulation of Th2 cytokines in DNA-TRYP(Pam)+MVA-TRYP immunization.

Twelve weeks after the final immunization, cells from individual spleens of immunized (DNA-TRYP+DNA-TRYP, DNA-TRYP+MVA-TRYP, DNA-TRYP(Pam)+MVA-TRYP) and control mice (DNA-pVAX(Pam)+MVA-Luc) were re-stimulated in vitro with recombinant TRYP or SLA for 72 hours followed by analysis of supernatants obtained for IFNγ, granzyme B, IL-13 and IL-10. Data are representative of 2 experiments; n = 3 mice/group. Mean±SE. *p<0.05, **p<.005.

Figure 2. TLR2 activation leads to increased CD4+ and CD8+ memory populations.

CD4 and CD8 T cell memory responses to TRYP antigen were examined 12 weeks after the MVA-TRYP boost A. Splenocytes from immunized and control groups of mice (3 mice/group) were CFSE labeled followed by in vitro stimulation with TRYP protein for 3 days to track proliferation of antigen specific cells. CD4 – Vector or DNA-TRYP+DNA-TRYP or DNA-TRYP+MVA-TRYP compared to DNA-TRYP(Pam)+MVA-TRYP is p<0.05. CD8 – Vector or DNA-TRYP+DNA-TRYP or DNA-TRYP+MVA-TRYP compared to DNA-TRYP(Pam)+MVA-TRYP is p<0.009. B. The cytokine responses of memory CD4 and CD8 cells in TRYP(Pam)+MVA-TRYP immunized mice (3 mice/group) were evaluated by FACS analysis of splenocytes stimulated in vitro with recombinant TRYP. Forward and side scatter were used to determine lymphocytes followed by gating on CD4+ or CD8+ cells. C. Overall frequency of antigen specific CD4 and CD8 cells producing IFNγ or IL-13 in different groups of vaccinated mice as determined by FACS analysis. Data are representative of two independent experiments. n = 3 mice/group.

Figure 3. TRYP(Pam3CSK4)-MVA-TRYP protects against L(V.) panamensis infection.

A. Lesion development in the control and groups of TRYP-immunized mice infected with 5×10⁴ L (V.) panamensis. n = 8/group For statistical significance comparison was made between DNA-TRYP(PAM3CSK4)+MVA-TRYP immunized mice and other groups of mice. B. Parasite burden at the site of infection and draining lymph node were determined by limiting dilution analysis at 6 weeks after infection. n = 4/group, * = p<0.05, ** = p<0.008. Results are representative of 2 independent experiments.

Figure 4. CD8 cells contribute to TLR1/2 mediated protection.

A. Lesion size (mean±SE) progression in L.(V).panamensis infected mice that were protectively immunized with DNA-TRYP(Pam)+MVA-TRYP and depleted of CD4 (-CD4) or CD8 cells (-CD8) prior to infection. ND, indicates not depleted or intact vaccinated mice. Controls include mice immunized vector plasmid and MVA. n = 10 mice per group. * p<0.05 Vector vs. DNA-TRYP(Pam)+MVA-TRYP; + p<0.05 DNA-TRYP(Pam)+MVA-TRYP CD4 depleted vs. DNA-TRYP(Pam)+MVA-TRYP; ++ p<0.005 DNA-TRYP(Pam)+MVA-TRYP CD4 depleted vs. DNA-TRYP(Pam)+MVA-TRYP; # p<0.005 Vector vs. DNA-TRYP(Pam)+MVA-TRYP CD8 depleted. B. Parasite burden (mean± SE) at the site of infection was determined by limiting dilution assay at 8 weeks after infection. (n = 4 mice per group). * p<0.05.

Figure 5. Modulation of cytokine production in TRYP(Pam)+MVA-TRYP protectively vaccinated mice depleted of CD8 or CD8 T cells.

At 8 weeks post-infection with L.(V).panamensis, the IFNγ, IL-13 or IL-10 responses were determined in DLNs of non-immunized and TRYP immunized mice (n = 4 per group). Cells from individual mice were pooled and then stimulated for 72 hours with SLA (in duplicate) and cytokine levels determined as indicated in the Materials and Methods Section. Mean±SD. *p<0.05, ** p<0.005; -CD4, CD4 depleted; -CD8, CD8 depleted; ND – Not Depleted.