BALB/c mice vaccinated with Leishmania major ribosomal proteins extracts combined with CpG oligodeoxynucleotides become resistant to disease caused by a secondary parasite challenge

Abstract

Leishmaniasis is an increasing public health problem and effective vaccines are not currently available. We have previously demonstrated that vaccination with ribosomal proteins extracts administered in combination of CpG oligodeoxynucleotides protects susceptible BALB/c mice against primary Leishmania major infection. Here, we evaluate the long-term immunity to secondary infection conferred by this vaccine. We show that vaccinated and infected BALB/c mice were able to control a secondary Leishmania major challenge, since no inflammation and very low number of parasites were observed in the site of reinfection. In addition, although an increment in the parasite burden was observed in the draining lymph nodes of the primary site of infection we did not detected inflammatory lesions at that site. Resistance against reinfection correlated to a predominant Th1 response against parasite antigens. Thus, cell cultures established from spleens and the draining lymph node of the secondary site of infection produced high levels of parasite specific IFN-gamma in the absence of IL-4 and IL-10 cytokine production. In addition, reinfected mice showed a high IgG2a/IgG1 ratio for anti-Leishmania antibodies. Our results suggest that ribosomal vaccine, which prevents pathology in a primary challenge, in combination with parasite persistence might be effective for long-term maintenance of immunity.

Figure 1

Course of L. major infection in BALB/c vaccinated mice. Mice (six per group) were s.c. immunized in the right footpad with three doses of the LRP adjuvated with CpG ODN (LRP + CpG), with the CpG ODN adjuvant (CpG) or with PBS. One month after the last immunization, the animals were infected in the left hind footpad with 5 × 10⁴L. major stationary phase promastigotes. (a) Footpad swelling is given as the difference of thickness between the infected and the uninfected contralateral footpad. (b) The number of viable parasites in the spleen and the popliteal DLN of the LRP + CpG ODN vaccinated were individually determined by limiting dilution at week eighteen post challenge. Results are expressed as the mean±SD of six spleens and popliteal DLN. (c) At week eighteen after footpad infection the level of IFN-γ, IL-10 and IL-4 was measure by ELISA in the supernatants of popliteal lymph node cells cultures from LRP + CpG ODN vaccinated mice. Cells were in vitro stimulated for 48 hours with 12 μg/ml of SLA or LRP and medium alone. Results are expressed as the mean±SD.

Figure 2

(a) Course of L. major infection in protected and reinfected BALB/c mice. Values represent the mean lesion diameter±standard deviation (SD). *P < .001: significant differences in inflammation for protected versus control mice at week seven postchallenge. (b) Seven weeks after reinfection, mice were euthanized and parasite burden in the ear dermis, spleen and in the retromaxillar DLN was individually quantitated. Results are expressed as the mean±SD of twelve ears and six spleens and DLN. *P < .001: significant decrease for reinfected versus control mice. (c) The parasite burden at week seven after rechallenge was individually quantitated in the popliteal lymph nodes of control and reinfected mice. Results are expressed as the mean±SD of six DLN. *P < .001: significant decrease for reinfected versus control mice.

Figure 3

Analysis of the cellular responses. At week seven after ear infection the level of IFN-γ (a), IL-10 (b), and IL-4 (c) was measured by ELISA in the supernatants of spleen and retromaxillar lymph node cells cultures from both mice groups. Cells were in vitro stimulated for 48 hours with 12 μg/ml of SLA or LRP and medium alone. Results are expressed as the mean±SD of twelve ears and DLN. (*P < .001).

Figure 4

Analysis of the humoral responses. Serum samples from control and vaccinated reinfected mice were obtained seven weeks after challenge in the ear dermis. The titre for IgG1 and IgG2a antibodies against LRP (a) and SLA (b) was determined individually by ELISA. Results are expressed as the mean±SD.