Protective immune responses against Schistosoma mansoni infection by immunization with functionally active gut-derived cysteine peptidases alone and in combination with glyceraldehyde 3-phosphate dehydrogenase

Abstract

Background: Schistosomiasis, a severe disease caused by parasites of the genus Schistosoma, is prevalent in 74 countries, affecting more than 250 million people, particularly children. We have previously shown that the Schistosoma mansoni gut-derived cysteine peptidase, cathepsin B1 (SmCB1), administered without adjuvant, elicits protection (>60%) against challenge infection of S. mansoni or S. haematobium in outbred, CD-1 mice. Here we compare the immunogenicity and protective potential of another gut-derived cysteine peptidase, S. mansoni cathepsin L3 (SmCL3), alone, and in combination with SmCB1. We also examined whether protective responses could be boosted by including a third non-peptidase schistosome secreted molecule, glyceraldehyde 3-phosphate dehydrogenase (SG3PDH), with the two peptidases.

Methodology/principal findings: While adjuvant-free SmCB1 and SmCL3 induced type 2 polarized responses in CD-1 outbred mice those elicited by SmCL3 were far weaker than those induced by SmCB1. Nevertheless, both cysteine peptidases evoked highly significant (P < 0.005) reduction in challenge worm burden (54-65%) as well as worm egg counts and viability. A combination of SmCL3 and SmCB1 did not induce significantly stronger immune responses or higher protection than that achieved using each peptidase alone. However, when the two peptidases were combined with SG3PDH the levels of protection against challenge S. mansoni infection reached 70-76% and were accompanied by highly significant (P < 0.005) decreases in worm egg counts and viability. Similarly, high levels of protection were achieved in hamsters immunized with the cysteine peptidase/SG3PDH-based vaccine.

Conclusions/significance: Gut-derived cysteine peptidases are highly protective against schistosome challenge infection when administered subcutaneously without adjuvant to outbred CD-1 mice and hamsters, and can also act to enhance the efficacy of other schistosome antigens, such as SG3PDH. This cysteine peptidase-based vaccine should now be advanced to experiments in non-human primates and, if shown promise, progressed to Phase 1 safety trials in humans.

Fig 1. Overview of vaccine schedule using SmCB1 and SmCL3 in mice.

Schedule shows regime of vaccine delivery and challenge infection with schistosome cercariae. Time-points of tissue sampling and parasitological analysis are also shown.

Fig 2. Overview of vaccine schedule using combination vaccine in mice and hamsters.

Schedule show regime of vaccine delivery and challenge infection with schistosome cercariae. Time-points of tissue sampling and parasitological analysis are also shown.

Fig 3. Serum antibody response to SmCB1 and SmCL3 following immunization and challenge infection of mice.

(A) Serum antibody reactivity to SmCB1 and SmCL3 was assessed by ELISA at 7 and 21 days after second immunization and 7 and 14 days following challenge infection with 150 cercariae of S. mansoni. (B) Antibody reactivity at 7 and 21 days after second immunization and 7 and 14 days after the challenge infection were compared at dilutions of 1:200 with each column denoting mean value for three mice +/- SD; asterisks indicate significance (* P < 0.05) of differences.

Fig 4. Cytokine response of SmCB1- and SmCL3- immunized mice to the immunogen.

Each column represents mean +/- SD cytokine levels in epidermal (A) and lymph node (B-D) cell cultures (three mice per group), 2 (epidermal cells, A) and 4 (LNC, B-D) days after infection. Asterisks indicate significance (* P < 0.05, ** P < 0.005) of differences between levels of cytokines released in cultures stimulated with 0 (medium) or 20 μg/ml immunogen.

Fig 5. Effect of immunization with SmCB1 and SmCL3 on S. mansoni egg development.

Percent immature (left box), mature (middle box) and dead (right box) ova was assessed seven weeks after infection with 150 S. mansoni cercariae in CD1 mice (6-8/group) that were unimmunized (left column in each box) or immunized with SmCB1 (middle column in each box) or SmCL3 (right column in each box). P values as assessed by the Mann-Whitney test; NS = not significant.

Fig 6. Immunogenicity of SmCL3 alone and in combination with SmCB1 and SG3PDH.

(A) Percent mice with serum IgG antibody binding to SmCL3 or SmCB1 significantly (P < 0.05) higher than naïve and 7-day infection mice is represented by grey and black columns, respectively (B & C). Ex vivo spleen cells from 3 mice from each group of naïve, 7-day infection, SmCL3-, SmCL3 + SmCB1-, or SmCL3+SmCB1+ SG3PDH (Antigen Mix)-immunized mice were stimulated 7 days after infection with S. mansoni with 20 μg/ml SmCL3 (grey columns) or SmCB1 (black columns) and spleen supernatants assayed for release of cytokines by capture ELISA. Each column represents percent mouse SC culture releasing cytokine detectable levels, higher than mean of naïve and controls + 2 SD.

Fig 7. Effect of immunization with SmCL3 alone or with SmCB and SG3PDH on parasitological parameters of challenge S. mansoni infection in outbred mice.

Mice (10 per group) unimmunized (Group 1), immunized with SmCL3 (Group 2), SmCL3 + SmCB1 (Group 3) or SmCL3 + SmCB1+ SG3PDH (Group 4) were challenged 3 weeks after second immunization with 120 cercariae of S. mansoni and assessed for parasitological parameters 6 weeks post infection. P values as assessed by the Mann-Whitney test (two-tailed). Reduction % = mean number in unimmunized mice–mean number in immunized mice/ mean number in unimmunized mice x 100.

Fig 8. Mouse serum antibody and cytokine response to SG3PDH delivered with cysteine peptidases.

Sera taken from control and immunized [SmCB1+ SG3PDH, SmCL3+ SG3PDH and SmCB1+SmCL3+ SG3PDH (Antigen Mix)] mice obtained 7 days after challenge S. mansoni infection were analyzed for antibody titers (A) and isotype (B) to SG3PDH in ELISA. Spleen cells obtained at the same time point were analyzed for cytokine responses following in vitro stimulation with 0 or 20 μg/ml SG3PDH (C). Each point or column represents mean values +/- SD for three mice per group. Asterisks indicate significance (* P < 0.05, ** P < 0.005) of differences between immunized & infected and control-infected mice.