The direct activation of the innate immune system by Toll-like receptor ligands represents a compelling strategy to improve the response to a broad spectrum of pathogens. In previous studies, we had shown that administration of CpG ODN type D/A reduced the severity of Leishmania
lesions when applied prior to infection and at the site of the challenge (25
). This report extends the previous study by showing that systemic administration of CpG ODN type D/A confers protection in both prophylactic and postexposure strategies. The protection conferred appears to be due to systemic effects arising from the activation of the immune system, as shown by reduced lesion severity upon challenge at a site distant from the site of CpG ODN treatment. Despite the milder pathology during the primary infection, macaques established an effective adaptive and anamnestic immune response and were protected from reinfection to a degree similar to that in untreated animals.
Several studies had shown that CpG ODN can be used in primates to improve the immune response to vaccines for hepatitis B, malaria, and L. major
). Indeed, clinical trials are under way to assess the safety of K-type ODN when administered together with a vaccine for hepatitis B. However, despite multiple studies with mice showing that CpG ODN are effective in prophylactic and postexposure strategies to prevent or ameliorate infection by a wide variety of pathogens (6
), the evidence that CpG ODN can act as immunoprotective agents in primates is, to our knowledge, limited to a single study of L. major
). As mentioned above, in that study, macaques received CpG ODN 3 days before and after the challenge at the site of infection. While important as a proof of concept, this form of treatment is impractical outside a research setting. The present study demonstrates that direct administration at the site of challenge is not required for the immunoprotective effects of CpG ODN. Indeed, systemic administration of CpG ODN (i.m. or s.c.) reduced the severity of the skin lesions to the same degree as in situ inoculation. This is unlikely to have been caused by CpG ODN directly reaching the forehead draining lymph nodes.
As in previous primate studies, the mechanism of protection against Leishmania
is not clear. Unlike murine leishmaniasis, where IL-12 and IFN-γ production are needed to direct a strong Th1 response that controls parasite growth and high levels of IL-12 have been associated with reduced lesion size (24
), in this study, the correlation between the IFN-γ responses in peripheral blood and the size of L. major
lesions in primates is less evident (2
). These data are consistent with a prior report by Gicheru et al. showing that vervet monkeys vaccinated with IL-12 and killed L. major
developed antigen-specific IFN-γ levels similar to those observed among convalescent monkeys but were not protected from infection (7
Although lesion size has been associated with parasite load, it is determined in large part by the inflammatory response mounted to the pathogen. Since CpG ODN type D/A induce pDC to secrete high levels of IFN-α, which stimulates monocytes to mature into active DC and activates NK cells to secrete IFN-γ, it is possible that these innate responses mediate the reduced lesion severity. This would explain why CpG ODN type K (which induces strong proinflammatory-cytokine and B-cell activation but low or no IFN-α) secretion failed to protect macaques from L. major
). Studies utilizing the newly developed type C CpG ODN, which induce “K-like” proinflammatory properties and “A-like” IFN-α secretion, may provide some insight into the role of IFN-α in CpG ODN-mediated protection against L. major
. It is possible, however, that changes in IFN-γ or IFN-α occurring immediately after treatment or locally at the site of infection would not be reflected in the frequency of antigen-specific IFN-γ-producing cells in peripheral blood weeks after infection. Further studies to assess whether the administration of CpG ODN results in activation of the innate immune system at the site of the challenge are being undertaken to better elucidate the mechanism of protection.
As suggested by murine studies, the time of administration of the CpG ODN relative to the challenge appears to influence the effect of the CpG ODN. Figures and suggest that while administration of CpG ODN consistently resulted in smaller lesions, macaques that received CpG ODN prior to infection had accelerated development of skin lesions, which peaked and resolved sooner than those in untreated macaques. In contrast, those that received CpG ODN in situ at the time of the challenge had delayed lesion development. This suggests that preadministration of the CpG ODN (shown to be critical in the control of rapidly dividing pathogens [15
]) preactivates the innate immune system, resulting in earlier inflammation at the site of the challenge. In contrast, CpG ODN administration at or after the time of the challenge appears to curtail lesion development (Fig. and ).
An important limitation in the use of CpG ODN as an immunoprotective agent was the apparent need to administer it before the time of infection. In this model, we have shown that type D/A CpG ODN can also function as therapeutic agents, diminishing the severity of established infections. Although results from animal studies cannot be directly extrapolated to human disease, these findings raise the possibility that systemic or intralesion administration of D/A ODN, alone or in combination with other antiparasitic agents, may accelerate the healing of the cutaneous leishmaniasis lesion in infected patients. Further studies will be needed to confirm this possibility.
There are an estimated 12 million cases of leishmaniasis worldwide. Despite numerous trials, leishmanization, the controlled induction of disease with a few live parasites, is the only successful prophylactic vaccination strategy employed so far (23
). However, this type of immunization has serious limitations, including the risk of developing full-blown disease, that have led most countries to stop the practice. Nonetheless, given its proven efficacy, leishmanization might be readopted in certain regions where leishmaniasis is endemic and applied on a broader scale if the size and duration of the cutaneous lesions could be moderated without compromising its ability to confer strong and durable immunity. Studies of mice had suggested that CpG ODN could be used in concert with leishmanization to improve the immune response and limit lesion development without sterilizing immunity (22
). The present study supports the idea that local administration of ODN might be of use to control the primary inoculation with live parasites without interfering with long-lasting protection. Of concern, upon reinfection, 4 of the 12 macaques that received CpG ODN systemically had high levels of parasites. However, this elevated parasite count was not associated with enlarged lesions. Together, these findings suggest that CpG ODN may be used to reduce the lesions induced during leishmanization, making it safer without loss of protective efficacy.