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. Author manuscript; available in PMC: 2011 Aug 4.
Published in final edited form as: Mem Inst Oswaldo Cruz. 2010 Aug;105(5):687–691. doi: 10.1590/s0074-02762010000500015

Immune responses to gp82 provide protection against mucosal Trypanosoma cruzi infection

Christopher S Eickhoff a, Olivia K Giddings a, Nobuko Yoshida c, Daniel F Hoft a,b,*
PMCID: PMC3150497  NIHMSID: NIHMS241783  PMID: 20835618

Summary

The potential use of the Trypanosoma cruzi metacyclic trypomastigote (MT) stage-specific molecule gp82 as a vaccine target has not been fully explored. We show that opsonization of T. cruzi MT with gp82-specific antibody prior to a relevant mucosal challenge significantly reduced parasite infectivity. We also investigated the immune responses as well as 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 after a biologically relevant oral parasite challenge.

Keywords: Trypanosoma cruzi, Mucosal Immunity, Vaccines

Introduction

Trypanosoma cruzi, the causative agent of Chagas’ disease, infects >11 million people and kills an estimated 50,000 individuals annually (CDC 2007). Up to 30% of infected individuals will develop complications from Chagas’ disease, which include cardiomyopathy and the mega-syndromes (megaesophagus and megacolon). Natural infection of humans occurs primarily through contact with T. cruzi contaminated reduviid excreta, initiating infection usually through breaks in the skin or through mucosal surfaces. Recent reports of large scale outbreaks following food or drink contamination demonstrate the importance of oral route T. cruzi infection (Alarcon et al. 2010; Beltrao et al. 2009; Dias et al. 2008; Nobrega et al. 2009; Pereira et al. 2009).

Because parasite proteins are expressed differentially in distinct T. cruzi life stages, it may be difficult to develop a vaccine that establishes sterile immunity. Glycoprotein-82 (gp82), a stage-specific protein present on the surface of T. cruzi metacyclic trypomastigotes (MT), has been shown to bind to gastric mucin and to facilitate Ca2+ signaling activity necessary for parasite internalization [reviewed in (Yoshida 2006) and (Yoshida 2009)]. Isolates of T. cruzi which lack gp82 expression are much less efficient in infecting mice orally than those which express high levels of gp82 (Cortez et al. 2003). Antibodies which bind to gp82 inhibit in vitro infection of epithelial cells (Neira et al. 2003). Although gp82 is only expressed by MT, it could be a good target for immune responses aimed at providing protection against initial immune invasion and intracellular replication cycles.

It is well documented that type 1 immune responses are critical for protection against both mucosal and systemic T. cruzi infection (Hoft et al. 2000; Hoft & Eickhoff 2005; Rodrigues et al. 2009). CpG motifs presented within ssDNA are known to induce type 1 immune responses mediated by toll-like receptor 9 (TLR9) stimulation, and have been used safely and effectively in mice and humans (Cooper et al. 2005; Dumais et al. 2002; Krieg 2000). Several independent groups have shown induction of protective immune responses against lethal T. cruzi challenges using CpG mixed with whole T. cruzi lysate or various recombinant T. cruzi proteins (Araujo et al. 2005; Frank et al. 2003; Hoft et al. 2007). In the current work, we investigate the possibility of developing a mucosal T. cruzi vaccine containing the gp82 protein. We show that intranasal vaccination with CpG + gp82 induces immune responses protective against mucosal T. cruzi challenge.

Materials and Methods

Parasites and Mice

Female Harlan Sprague Dawley BALB/c mice aged 6 – 8 weeks were used throughout these studies and housed in AAALAC accredited facilities. Tulahuén strain T. cruzi insect-derived MT (IMT), culture-derived MT (CMT) and blood form trypomastigotes (BFT) were prepared as previously described (Giddings et al. 2006; Hoft & Eickhoff 2005).

Opsonization and T. cruzi challenges

IMT mixed 1:1 with 0.5 mg/ml MAb-3F6 (gp82-specific monoclonal antibody) or isotype control (Sigma, St. Louis, MO) were incubated at room temperature for 30 minutes prior to conjunctival or oral challenges done as previously described (Giddings et al. 2006; Hoft 1996).

Immunization with CpG-gp82

Recombinant gp82 and control GST proteins were prepared as described previously (Santori et al. 1996). Oligodeoxynucleotide (ODN) # 1826 (5’-TCCATGACGTTCCTGACGTT-3’) containing CpG motifs (underlined) and control ODN were purchased from Coley Pharmaceuticals group (Wellesley, MA). BALB/c mice were immunized intranasally twice, two weeks apart using 10 μg CpG 1826 mixed with 2 – 10 μg gp82 (or control GST).

Measurement of vaccine induced immunity

Four weeks after immunization, spleen cells from representative vaccinated mice were stimulated at 1 × 106 cells/ml in round-bottom 96 well plates with 0.4 – 2.0 μg/ml recombinant gp82 or control GST protein. Spleen cells (4 × 106/ml) from these same immunized mice were stimulated in 24 well plates with medium or 10 μg/ml T. cruzi lysate (Hoft & Eickhoff 2005). Three days later proliferation and IFN-γ secretion were assessed as previously described (Hoft & Eickhoff 2005).

Determination of parasite infectivity

Four weeks after the final immunization, some mice (N = 5/group) were challenged with 5,000 BFT s.c. and survival monitored as a means of analyzing systemic protective immunity. Other groups of mice (N = 5 to 8/group) were challenged orally with 1,000 IMT and parasite replication in mucosal tissues determined using T. cruzi-specific real-time PCR. Mice were harvested 11–13 days after mucosal T. cruzi challenge and levels of parasite replication determined in draining lymph node and tissue DNA samples using T. cruzi-specific real-time PCR as previously described (Giddings et al. 2006; Hoft et al. 2007). Briefly, DNA was purified from splenic and gastric lymph node tissue (after oral MT challenge) and from submandibular lymph nodes (after conjunctival MT challenge) using a commercially available kit (DNeasy, QIAGEN). T. cruzi specific real-time PCR was performed used using 100 ng purified DNA per reaction.

Ethics

All animal studies were performed in Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) accredited facilities (NIH Assurance number A3225-01). In addition, all mouse studies were approved by the Institutional Animal Care and Use Committee (IACUC)/Animal Care Committee (ACC) at Saint Louis University.

Results

Gp82-specific antibodies reduce T. cruzi infectivity

Our group has shown that opsonization of T. cruzi MT with fecal extracts containing T. cruzi-specific secretory IgA can reduce parasite infectivity after conjunctival challenge (Giddings et al. 2006). In the present work, we incubated Tulahuén strain IMT with a control or gp82-specific monoclonal antibody (MAb-3F6). Opsonized parasites were then placed on the conjunctiva of anesthetized mice, or fed orally to naïve mice. Two weeks later, mice were sacrificed and parasite replication determined by real-time PCR in tissues taken from sites of early infection. As seen in Fig. 1a, mice challenged conjunctivally with anti-gp82 opsonized parasites had significantly reduced parasite DNA detectible in the lymph nodes draining the site of initial invasion in the nasal cavity compared with mice infected with control opsonized parasites (p<0.04 by Mann-Whitney U test). Similarly, mice challenged orally with anti-gp82 opsonized parasites had ~4 fold lower amounts of T. cruzi DNA recoverable at the site of initial invasion in gastric tissue compared with controls (Fig. 1b). These results show that gp82 specific antibodies can reduce infectivity at the initial sites of parasite mucosal invasion after both conjunctival and oral T. cruzi challenges. In addition, significantly decreased amounts of parasite DNA were detected in splenic DNA samples isolated from mice challenged orally with anti-gp82 opsonized MT compared with mice challenged with control opsonized MT (Fig. 1c., p<0.02 by Mann-Whitney U test). Thus, systemic spread of parasite infection can be curtailed by opsonization with gp82-specific antibody, suggesting that overall T. cruzi disseminated disease could be limited by neutralization of initial parasite infectivity.

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

Figure 1

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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).

Intranasal vaccination with CpG-gp82 induces type 1 T. cruzi-specific immune responses

Systemic immunization with alum + gp82 has previously been shown to induce immune responses which were capable of reducing parasitemia after systemic MT challenge (Santori et al. 1996). In the work presented here, we intranasally vaccinated mice with gp82 adjuvanted with toll like receptor 9 signaling CpG. Four weeks following the second and final immunization, spleen cells from immunized animals were harvested and stimulated with antigen. Spleen cells from mice immunized with CpG-gp82 proliferated in a dose dependant manner in response to recombinant gp82 antigen stimulation in vitro (Fig. 2a). Spleen cells from these same vaccinated animals produced IFN-γ upon in vitro stimulation with gp82 (Fig. 2b). Additionally, spleen cells from mice immunized with CpG-gp82 produced IFN-γ after in vitro stimulation with whole T. cruzi lysate (Fig. 2c). The production of IFN-γ by spleen cells from gp82 immunized mice after stimulation with T. cruzi antigens is especially important, since type 1 immune responses have been shown to be critical for protection against both systemic and mucosal T. cruzi challenges (Hoft & Eickhoff 2002; Hoft & Eickhoff 2005).

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

Figure 2

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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.

Intranasal CpG-gp82 immunization fails to protect mice against systemic BFT challenge

In order to assess protective immunity induced by CpG-gp82 immunization, groups of immunized mice were challenged systemically or mucosally four weeks after their second and final vaccination. For systemic survival studies, 5,000 BFT diluted in 100μl PBS were injected subcutaneously at the base of the tail. As seen in Fig. 3a, mice immunized with CpG-gp82 failed to survive lethal T. cruzi challenge, and time to death was not prolonged.

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

Figure 3

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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).

CpG-gp82 immunization results in protection after a biologically relevant mucosal MT challenge

In order to study mucosal protection, immunized mice were challenged orally with 1,000 IMT. DNA was extracted from stomachs 11 – 13 days after oral challenge and infection levels measured using T. cruzi specific real-time PCR. Gastric DNA obtained from CpG-gp82 vaccinated mice contained significantly reduced amounts of T. cruzi DNA compared with gastric DNA samples from CpG-negative control GST immunized mice (Fig. 3b, p<0.05 by Mann-Whitney U test).

Discussion

All four T. cruzi life stages have been studied for gp82 expression by simple IFA, western blot, and more recently by proteome analysis (CDC, 2007; Atwood, III et al. 2005; Teixeira & Yoshida 1986). It is clear that gp82 is only expressed by T. cruzi MT, and not by T. cruzi intracellular amastigotes or BFT. The critical role of gp82 in parasite mucosal epithelial invasion makes this T. cruzi glycoprotein an ideal target for mucosal immune responses. It is a highly conserved molecule present in nearly all T. cruzi isolates including strains Y, CL, F, Tulahuén, G and others (Yoshida 2006). Epimastigotes do not express gp82 and are non-infectious (Manque et al. 2003). In addition, gp82 deficient MT are deficient in mucosal infectivity in mice after oral delivery (Cortez et al. 2003). The binding of gp82 to gastric mucin induces intracellular Ca+ increases dependent on ATP consumption, which appear to be required for parasite internalization. The interaction / binding of gp82 to gastric mucin may explain earlier findings in which MT, but not BFT, efficiently infect mice after oral challenge (Hoft 1996). Previous work has shown that antibodies directed against gp82 significantly reduce parasite infectivity both in vitro and after oral in vivo challenges. We have extended these findings by 1) showing that anti-gp82 Abs can block T. cruzi infection via both conjunctival and oral routes, and 2) demonstrating that a mucosal vaccine can significantly reduce mucosal infection after oral T. cruzi challenge. It seems likely that the gp82 gastric mucin binding epitope overlaps the gp82-specific MAb-3F6 epitope. Alternatively, immune responses directed against gp82 could block the induction of Ca+/ATP mediated membrane repair responses, thereby preventing active parasite internalization.

It has been reported that mice challenged systemically with MT after gp82 vaccination develop lower parasitemia compared with control immunized mice (Santori et al. 1996). However, those data were generated using an MT challenge as opposed to BFT challenge, and analyzed peak parasitemia rather than death. In our work we found no evidence for protection against systemic BFT challenge. Stage-specific gp82 expression on MT (not AMA or BFT) is the most logical explanation for these apparently conflicting data, as immune responses directed towards MT may not be effective when the MT life stage is bypassed. Because the Santori et al. group challenged with MT, the gp82-specific immunity induced could have partially protected against this initial infecting stage. However, we challenged systemically with BFT not expressing gp82 and therefore the gp82-specific immunity is likely to have been irrelevant for recognition of this infection.

Many publications investigating T. cruzi vaccine development have focused on the induction of systemic immune responses and utilize lethal but perhaps biologically irrelevant challenge models. As new natural infections of humans occur through contact with IMT contaminated reduviid excreta, we must investigate the effects of vaccine-induced immunity on these types of infections. Our results here which show gp82 vaccine-induced mucosal protection after a biologically relevant IMT challenge are one such example of these types of necessary studies. Future T. cruzi vaccines should encode antigens that can induce both mucosal and systemic immunity. The gp82 antigen could facilitate the induction of optimal mucosal protection, but other antigens will be required for induction of optimal systemic protection.

Prevalence of human T. cruzi infection increases drastically with the presence of infected dogs in households (Cohen & Gurtler 2001). Removal of infected dog reservoirs alone can reduce the threat of human T. cruzi transmission. Previous studies of dogs immunized with live-attenuated T. cruzi epimastigotes provided partial protection against natural T. cruzi infection (Basombrio et al. 1993). At the present time, it may be more feasible to develop a vaccine aimed at preventing systemic and mucosal infection of domesticated animals rather than human beings. Our data clearly show that immunization with gp82 induces significant mucosal protection and could be useful in vaccines designed to prevent mucosal infection in dogs.

Other vaccines composed of T. cruzi proteins expressed by BFT and/or AMA (including TS and ASP-2) induce strong T cell responses and systemic protection in mice (Araujo et al. 2005; Fujimura et al. 2001). A vaccine composed of several stage-specific proteins could perhaps lead to optimal protective immunity by inducing immune responses relevant for both mucosal and systemic protection. Focusing future vaccine strategies on domestic animals would be much safer than vaccinating humans, and could interrupt transmission in households. Thus a multi-component vaccine including gp82 aimed at preventing T. cruzi infection in dogs should be explored.

Acknowledgments

We wish to thank Vanessa D. Atayde for preparation and purification of recombinant gp82.

Sponsorships: This work was supported by the National Institutes of Health grant RO1 AI040196 to D.F.H and from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) to N.Y.

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