IMMUNOLOGY AND HOST-PARASITE INTERACTIONS - LETTER TO THE EDITOR Adjuvant-free schistosome cathepsin L3 is an efficacious schistosomiasis vaccine–comment on Huang et al.: Characteristics and function of cathepsin L3 from Schistosoma japonicum Hatem Tallima 1 & Rashika El Ridi 1 Received: 2 April 2020 /Accepted: 28 May 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020 Dear Editor: We read with interest the article by Huang et al. (2020) reporting on the Schistosoma japonicum cathepsin L3 gene successful cloning and expression, accurate protein distribu- tion in the different parasites stages, especially the adult mus- culature and alimentary canal, and protective capacity against challenge infection. We were nevertheless appalled by the last sentence in the Discussion stating that “SjCL3 is not suitable as an antigen for developing an anti-schistosome vaccine,” and herein show the reasons that led to this incorrect statement. Misleading enzymatic activity assay The principle of the enzymatic assay based on L-tyrosine re- lease and “contrast” control was not explained, standardized, and verified. The standard fluorometric substrate assay (Kuhelj et al. 1995; Collins et al. 2004; Dvorák et al. 2009) was used to assess the proteolytic activity of Fasciola hepatica cathepsin L1, FhCL1 (El Ridi et al. 2014 ), Schistosoma mansoni cathepsin L3 (Dvorák et al. 2009; Tallima et al. 2017a), and Schistosoma haematobium cathep- sin L, ShCL (Abdel Aziz et al. 2019). Accordingly, the enzy- matic activity of SjCL3 may not be compared with that of the schistosome cathepsins L shown to be promising vaccine can- didates. In fact, none of three recombinant S. mansoni cathep- sins B and L previously expressed, similarly to SjCL3, in Escherichia coli exhibited detectable proteolytic activity. Absence of activity was expected as these enzymes possess many cysteine residues which have difficulty in refolding within the reducing milieu of the E. coli system in order to form six disulfide bonds (Dolinar et al. 1995; Dalton et al. 1996; Dvorák et al. 2009). Schistosoma haematobium ShCL was expressed in E. coli and found to lack enzymatic activity (Abdel Aziz et al. 2019). Enzymatically inactive papain (Tallima et al. 2019), FhCL1 (El Ridi et al. 2014), and ShCL (Abdel Aziz et al. 2019) all induced highly significant (up to P < 0.0005) reduction in challenge worm burdens. The per- cent reduction was, however, clearly lower than that achieved using the active enzymes. The moderate, yet significant, pro- tection obtained with SjCL3, justifying the last sentence, could simply be due to a questionable enzymatic potency. Inappropriate adjuvant use Papain and helminth cathepsins were proposed as vaccine candidates based on their inbuilt ability to skew the immune responses towards the type 2 axis, leading to generation of preponderant type 2 cytokines and related antibody isotypes (El Ridi et al. 2014). Accordingly, these cysteine peptidases were used for protection against schistosome infection with- out any adjuvant, as they possess and display inbuilt adjuvanticity (El Ridi et al. 2014; Tallima et al. 2015, 2017a, 2017b; Abdel Aziz et al. 2019). Use of the type 1 immunity- inducing complete Freund’s adjuvant (CFA) would powerful- ly antagonize the action and role of the cysteine peptidase immunogen. Nevertheless, with the reported standard devia- tion values, the significance of differences in worm burdens between SjCL3 and adjuvant groups (Huang et al. 2020, Figure 4) would be in the level of P < 0.0004, if five mice were used, and P < 0.0001, if the standard 10 mice per group were used. The authors failed to emphasize this finding, which is far more meaningful and reliable than the percentage Section Editor: Una Ryan * Rashika El Ridi rashika@sci.cu.edu.eg; rashikaelridi@gmail.com 1 Zoology Department, Faculty of Science, Cairo University, Giza 12613, Egypt Parasitology Research https://doi.org/10.1007/s00436-020-06737-w