Immune responses to gp82 provide protection against mucosal Trypanosoma cruzi infection

Abstract

The potential use of the Trypanosoma cruzi metacyclic trypomastigote (MT) stage-specific molecule glycoprotein-82 (gp82) as a vaccine target has not been fully explored. We show that the opsonization of T. cruzi MT with gp82-specific antibody prior to mucosal challenge significantly reduces parasite infectivity. In addition, we investigated the immune responses as well as the systemic and mucosal protective immunity induced by intranasal CpG-adjuvanted gp82 vaccination. Spleen cells from mice immunized with CpG-gp82 proliferated and secreted IFN-γ in a dose-dependent manner in response to in vitro stimulation with gp82 and parasite lysate. More importantly, these CpG-gp82-immunized mice were significantly protected from a biologically relevant oral parasite challenge.

Figure 1. Decreased parasite infectivity after opsonization with gp82-specific antibodies

Insect-derived metacyclic trypomastigotes (IMT) were incubated with control or anti-gp82 specific monoclonal antibodies and then either placed on the conjunctiva of anesthetized mice (panel a) or fed orally to BALB/c mice (panels b and c). DNA extracted 11–13 days later from local draining tissues (parotid and submandibular lymph nodes from conjunctivally challenged mice or gastric DNA from orally challenged animals) or distant sites (spleen) were used in T. cruzi specific real-time PCR assays. In panel a, the numbers of T. cruzi molecular equivalents per 100ng of DNA is significantly lower (p < 0.04 by Mann-Whitney U Test) in anti-gp82 opsonized MT-infected mice than in control opsonized MT-infected mice. Similarly, mice challenged orally had markedly reduced amounts of T. cruzi DNA present in gastric samples after opsonization with anti-gp82 MAb-3F6 as compared to controls (panel b). In addition, systemic spread of parasites was inhibited by opsonization with anti-gp82, as seen in panel c where reduced parasite DNA was detectable in spleens recovered from mice that were infected with anti-gp82 MAb 3F6-opsonized MT with compared with control opsonized MT (p < 0.02 Mann-Whitney U Test).

Figure 2. Type 1 mediated immune responses induced by intranasal CpG-gp82 immunization

BALB/c mice were immunized twice two weeks apart with 10μg CpG 1826 mixed with 2.5–10 μg gp82 or control GST protein. Four weeks later, spleen cells were removed and stimulated in vitro for 3 days with control protein, recombinant gp82, or T. cruzi lysate, after which time proliferative responses were detected using ³H-thymidine incorporation and IFN-γ secretion measured via ELISA. Negative control stimulation values were subtracted. Spleen cells from mice immunized with CpG-gp82 proliferated and produced IFN-γ in a dose dependent manner in response to recombinant gp82 stimulation in vitro (a and b). As seen in panel c, mice immunized with CpG-gp82 also produced IFN-γ in response to T. cruzi lysate stimulation. Based on these immune response results, 10μg gp82 was chosen for immunization and challenge experiments to assess vaccine-induced protective immunity.

Figure 3. Mucosal protective immunity induced by intranasal CpG-gp82 immunization

BALB/c mice were immunized intranasally twice two weeks apart with 10μg CpG 1826 mixed with 10 μg gp82 or control GST protein. Four weeks after the final immunization, groups of mice were challenged with 5,000 BFT s.c. (a) or with 1,000 IMT PO (b). Control and CpG-gp82 immunized mice failed to survive challenge with a normally lethal dose of T. cruzi BFT (panel a). However, as compared with the control immunized group, mice vaccinated with CpG-gp82 had significantly lower amounts of T. cruzi DNA detectable in gastric DNA samples taken 12 days post infection as determined by T. cruzi specific real-time PCR (panel b, p < 0.05 using Mann-Whitney U test).