Natural killer T (NKT) cells are a unique population of lymphocytes that coexpress a semiinvariant T cell and natural killer cell receptors, which are particularly abundant in the liver. To investigate the possible effect of these cells on the development of the liver stages of malaria parasites, a glycolipid, α-galactosylceramide (α-GalCer), known to selectively activate Vα14 NKT cells in the context of CD1d molecules, was administered to sporozoite-inoculated mice. The administration of α-GalCer resulted in rapid, strong antimalaria activity, inhibiting the development of the intrahepatocytic stages of the rodent malaria parasitesPlasmodium yoeliiandPlasmodium berghei. The antimalaria activity mediated by α-GalCer is stage-specific, since the course of blood-stage-induced infection was not inhibited by administration of this glycolipid. Furthermore, it was determined that IFN-γ is essential for the antimalaria activity mediated by the glycolipid. Taken together, our results provide the clear evidence that NKT cells can mediate protection against an intracellular microbial infection.

Brucella abortus is a facultative intracellular bacterium that infects humans and domestic animals. The enhanced susceptibility to virulent B. abortus observed in MyD88 knockout (KO) mice led us to investigate the mechanisms involved in MyD88-dependent immune responses. First, we defined the role of MyD88 in dendritic cell (DC) maturation. In vitro as well as in vivo, B. abortus-exposed MyD88 KO DCs displayed a significant impairment on maturation as observed by expression of CD40, CD86, and MHC class II on CD11c⁺ cells. In addition, IL-12 and TNF-α production was totally abrogated in MyD88 KO DCs and macrophages. Furthermore, B. abortus-induced IL-12 production was found to be dependent on TLR2 in DC, but independent on TLR2 and TLR4 in macrophages. Additionally, we investigated the role of exogenous IL-12 and TNF-α administration on MyD88 KO control of B. abortus infection. Importantly, IL-12, but not TNF-α, was able to partially rescue host susceptibility in MyD88 KO-infected animals. Furthermore, we demonstrated the role played by TLR9 during virulent B. abortus infection. TLR9 KO-infected mice showed 1 log Brucella CFU higher than wild-type mice. Macrophages and DC from TLR9 KO mice showed reduced IL-12 and unaltered TNF-α production when these cells were stimulated with Brucella. Together, these results suggest that susceptibility of MyD88 KO mice to B. abortus is due to impaired DC maturation and lack of IL-12 synthesis. Additionally, DC activation during Brucella infection plays an important regulatory role by stimulating and programming T cells to produce IFN-γ.

Initial host defense to bacterial infection is executed by innate immunity, and therefore the main goal of this study was to examine the contribution of Toll-like receptors (TLRs) duringBrucella abortusinfection. CHO reporter cell lines transfected with CD14 and TLRs showed thatB. abortustriggers both TLR2 and TLR4. In contrast, lipopolysaccharide (LPS) and lipid A derived fromBrucellarough (R) and smooth (S) strains activate CHO cells only through TLR4. Consistently, macrophages from C3H/HePas mice exposed to R and S strains and their LPS produced higher levels of tumor necrosis factor alpha (TNF-α) and interleukin-12 compared to C3H/HeJ, a TLR4 mutant mouse. The essential role of TLR4 for induction of proinflammatory cytokines was confirmed with diphosphoryl lipid A fromRhodobacter sphaeroides. Furthermore, to determine the contribution of TLR2 and TLR4 in bacterial clearance, numbers ofBrucellawere monitored in the spleen of C3H/HeJ, C3H/HePas, TLR2 knockout, and wild-type mice at 1, 3, and 6 weeks followingB. abortusinfection. Interestingly, murine brucellosis was markedly exacerbated at weeks 3 and 6 after infection in animals that lacked functional TLR4 (C3H/HeJ) compared to C3H/HePas that paralleled the reduced gamma interferon production by this mouse strain. Finally, by mass spectrometry analysis we found dramatic differences on the lipid A profiles of R and S strains. In fact, S lipid A was shown to be more active to trigger TLR4 than R lipid A in CHO cells and more effective in inducing dendritic cell maturation. In conclusion, these results indicate that TLR4 plays a role in resistance toB. abortusinfection and that S lipid A has potent adjuvant activity.

We report that complete protection against malaria and total inhibition of liver stage development and parasitemia was obtained in 100% of BALB/c mice primed with a replication-defective recombinant adenovirus expressing the circumsporozoite (CS) protein ofPlasmodium yoelii(AdPyCS), followed by a booster with an attenuated recombinant vaccinia virus, expressing the same malaria antigen, VacPyCS. We found increased levels of activated CS-specific CD8⁺and CD4⁺T cells, higher anti-sporozoite antibody titers, and greater protection in these mice, when the time between priming and boosting with these two viral vectors was extended from 2 to 8 or more weeks. Most importantly, by using this immunization regimen, the protection of the immunized mice was found to be long-lasting, namely complete resistance to infection of all animals 3 1/2 months after priming. These results indicate that immunization with AdPyCS generates highly effective memory T and B cells that can be recalled long after priming by boosting with VacPyCS.

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is a prototypical neglected tropical disease. Specific immunity promotes acute phase survival. Nevertheless, one-third of CD patients develop chronic chagasic cardiomyopathy (CCC) associated with parasite persistence and immunological unbalance. Currently, the therapeutic management of patients only mitigates CCC symptoms. Therefore, a vaccine arises as an alternative to stimulate protective immunity and thereby prevent, delay progression and even reverse CCC. We examined this hypothesis by vaccinating mice with replication-defective human Type 5 recombinant adenoviruses (rAd) carrying sequences of amastigote surface protein-2 (rAdASP2) and trans-sialidase (rAdTS) T. cruzi antigens. For prophylactic vaccination, naïve C57BL/6 mice were immunized with rAdASP2+rAdTS (rAdVax) using a homologous prime/boost protocol before challenge with the Colombian strain. For therapeutic vaccination, rAdVax administration was initiated at 120 days post-infection (dpi), when mice were afflicted by CCC. Mice were analyzed for electrical abnormalities, immune response and cardiac parasitism and tissue damage. Prophylactic immunization with rAdVax induced antibodies and H-2K^b-restricted cytotoxic and interferon (IFN)γ-producing CD8⁺ T-cells, reduced acute heart parasitism and electrical abnormalities in the chronic phase. Therapeutic vaccination increased survival and reduced electrical abnormalities after the prime (analysis at 160 dpi) and the boost (analysis at 180 and 230 dpi). Post-therapy mice exhibited less heart injury and electrical abnormalities compared with pre-therapy mice. rAdVax therapeutic vaccination preserved specific IFNγ-mediated immunity but reduced the response to polyclonal stimuli (anti-CD3 plus anti-CD28), CD107a⁺ CD8⁺ T-cell frequency and plasma nitric oxide (NO) levels. Moreover, therapeutic rAdVax reshaped immunity in the heart tissue as reduced the number of perforin+ cells, preserved the number of IFNγ⁺ cells, increased the expression of IFNγ mRNA but reduced inducible NO synthase mRNA. Vaccine-based immunostimulation with rAd might offer a rational alternative for re-programming the immune response to preserve and, moreover, recover tissue injury in Chagas’ heart disease. The idea that Chagas disease (CD) has an important autoimmune involvement contributed to delay the development of therapies and vaccines. CD is a parasitic neglected disease which afflicts millions of people mostly in Latin America. The cardiac form is the main clinical manifestation of CD. Currently, patients with access to therapy receive medicaments that only mitigate symptoms. Because of the limited prospect of treatment, vaccine reemerged as a strategy to prevent infection, interfere with CD progression and, moreover, reverse heart abnormalities. Here we tested a recombinant adenovirus carrying sequences of ASP2 and TS T. cruzi antigens (rAdVax) as prophylactic and therapeutic tool using a model of chronic Chagas’ heart disease. We showed that prophylactic vaccination reduced heart parasite load, inflammation and electrical abnormalities. The rAdVax therapeutic vaccination also reduced heart injury and improved electrical function, preserved specific IFNγ-mediated immunity but reduced response to polyclonal stimuli, CD107a⁺ CD8⁺ T-cell frequency and plasma nitric oxide levels. Moreover, therapeutic rAdVax preserved the number IFNγ⁺ cells, but decreased perforin⁺ cells in the heart tissue. Therefore, our results support the hypothesis that vaccination can modify the immunological unbalance that concurs to Chagas’ heart disease to improve prognosis.