The immunodominant T helper 2 (Th2) response elicited in BALB/c mice by the Leishmania LiP2a and LiP2b acidic ribosomal proteins cannot be reverted by strong Th1 inducers

Abstract

The search for disease-associated T helper 2 (Th2) Leishmania antigens and the induction of a Th1 immune response to them using defined vaccination protocols is a potential strategy to induce protection against Leishmania infection. Leishmania infantum LiP2a and LiP2b acidic ribosomal protein (P proteins) have been described as prominent antigens during human and canine visceral leishmaniasis. In this study we demonstrate that BALB/c mice infected with Leishmania major develop a Th2-like humoral response against Leishmania LiP2a and LiP2b proteins and that the same response is induced in BALB/c mice when the parasite P proteins are immunized as recombinant molecules without adjuvant. The genetic immunization of BALB/c mice with eukaryotic expression plasmids coding for these proteins was unable to redirect the Th2-like response induced by these antigens, and only the co-administration of the recombinant P proteins with CpG oligodeoxynucleotides (CpG ODN) promoted a mixed Th1/Th2 immune response. According to the preponderance of a Th2 or mixed Th1/Th2 responses elicited by the different regimens of immunization tested, no evidence of protection was observed in mice after challenge with L. major. Although alterations of the clinical outcome were not detected in mice presensitized with the P proteins, the enhanced IgG1 and interleukin (IL)-4 response against total Leishmania antigens in these mice may indicate an exacerbation of the disease.

Fig. 1

(a) Purification of Leishmania infantum rLiP2a and rLiP2b after expression in Escherichia coli. Five μg of rLiP2a (lane 1) and rLiP2b (lane 2) were electrophoresed on linear 10–14% gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel. The Coomassie-blue staining of the gel is shown. (b–d) Heterologous cytoplasmic expression of Leishmania P proteins in COS-7 cells. Immunofluorescence of COS-7 cells transfected transiently with pcDNA3-LiP2a (b) and pcDNA3-LiP2b (c); 24 h after transfection cells were fixed and immunostained with a polyclonal anti-LiP2a (b) or anti-LiP2b serum (c). As secondary antibody an Alexa488-conjugated goat anti-mouse IgG was employed. As a control, COS-7 cells transfected with pcDNA3-LiP2a were incubated with the sera from preimmune mice (d). Similar results were obtained when cells transfected with pcDNA-LiP2b were incubated with the corresponding preimmune sera (data not shown).

Fig. 2

(a) Leishmania P proteins induced a predominant T helper 2-like response in L. major-infected BALB/c mice. Analysis of the anti-LiP2a and anti-LiP2b humoral response in BALB/c mice infected with L. major. Six naive BALB/c mice were inoculated with 5 × 10⁴ stationary-phase L. major promastigotes into the left footpad. Sera were obtained after 8 weeks of infection and tested individually by enzyme-linked immunosorbent assay (ELISA) against the recombinant rLiP2a and rLiP2b P proteins. Reciprocal end-point titres for IgG1 and IgG2a antibodies subclasses were determined as indicated in Materials and methods. (b) Analysis of the specific humoral response induced in BALB/c mice immunized with the LiP2a and LiP2b acidic ribosomal proteins without adjuvants. Six mice were immunized twice in the right footpad with 2 μg of rLiP2a or rLiP2b. Sera were obtained and assayed individually by ELISA 7 days after the first immunization (white bars) or 7 days after the second immunization (solid bars). Reciprocal end-point titre was determined for IgM, IgG, IgG1 and IgG2a antibodies subclasses. No reactivity was detected in the preimmune sera. (c–d) Immune responses elicited by the genetic inoculation of parasite P proteins. BALB/c mice (six mice per group) were immunized with the recombinant rLiP2a or rLiP2b proteins alone and adjuvated with CpG oligodeoxynucleotides (ODN), by genetic vaccination and by prime-boosting regimen, as indicated in Materials and methods. As control, mice were vaccinated in the same manner with phosphate-bufered saline, with CpG ODN alone, with the empty pcDNA3 plasmid and with the empty plasmid and CpG ODN. For measurement of the specific anti-LiP2a (b) or anti-LiP2b (c) humoral responses sera were individually assayed by ELISA 7 days after the last immunization. The reciprocal end-point titre is shown. None of the preimmune or the control sera showed reactivity against the corresponding antigens.

Fig. 3

Interferon (IFN)-γ production by splenocytes of BALB/c mice stimulated in vitro with the rLiP2a (a) and rLiP2b (b) P proteins. Groups of mice described in Fig. 2 were killed 4 weeks after the last immunization, and their splenocytes were obtained and cultured in vitro for 48 h in the presence of the corresponding rLiP2a or rLiP2b soluble proteins or medium alone. The supernatants were harvested and assayed individually for IFN-γ production by enzyme-linked immunosorbent assay.

Fig. 4

Course of Leishmania major infection in BALB/c vaccinated mice. Mice (six per group) were immunized twice s. c. with the recombinant rLiP2a or rLiP2b proteins adjuvated with CpG oligodeoxynucleotides (ODN) (a), primed twice with the recombinant plasmid and boosted with the rLiP2a or rLiP2b adjuvated with CpG ODN (b), or three times with the pcDNA3-LiP2a or pcDNA3-LiP2b eukaryotic expression plasmids (c), as indicated in Materials and methods. As control, mice were vaccinated in the same manner with CpG ODN alone (a), with the empty plasmid and CpG ODN (b) and with the empty pcDNA3 plasmid (c). For comparison, unvaccinated groups injected with phosphate-buffered saline were included (a–c). One month after the last immunization, mice were infected in the left hind footpad with 5 × 10⁴ L. major stationary-phase promastigotes. Footpad swelling is given as the difference of thickness between the infected and the uninfected contralateral footpad. No significant differences were found between vaccinated mice and their corresponding controls in any group.

Fig. 5

Analysis of the immune response against the acidic ribosomal proteins in vaccinated and control mice after Leishmania major challenge. Serum samples from different groups of vaccinated and control mice were obtained 7 weeks after challenge and the reciprocal end-point titres for IgG1 and IgG2a antibodies against rLiP2a (a) and rLiP2b (b) were determined by enzyme-linked immunosorbent assay (ELISA). At the same time mice were killed and their splenocytes were obtained and cultured in vitro in complete medium with or without the recombinant proteins. The supernatants were harvested and the specific rLiP2a-driven (c) or rLiP2b-driven (d) production of interferon-γ, and the specific rLiP2a-driven (e) or rLiP2b-driven (f) production of interleukin-4 was assayed by ELISA.

Fig. 6

Analysis of the immune response against total parasite proteins induced by the Leishmania major challenge in vaccinated and control mice. (a) Analysis of the humoral response against soluble Leishmania antigen (SLA) from the different regimens of vaccination and the corresponding controls. Serum samples were obtained 7 weeks after challenge and the reciprocal end-point titre for IgG1 and IgG2a antibodies against SLA was determined individually by enzyme-linked immunosorbent assay (ELISA). The ratio between IgG1 and IgG2a titres for the different groups has been also included. Seven weeks after infection animals from the different regimens of vaccination and the corresponding controls were killed and their splenocytes were obtained and cultured in vitro in complete medium with or without SLA. The supernatants were harvested and assayed for interferon-γ (b) and interleukin-4 (c) by ELISA. *P < 0·05; **P < 0·02.