Cross-protection induced by Toxoplasma gondii virus-like particle vaccine upon intraperitoneal route challenge

Acta Tropica 164 (2016) 77–83

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Cross-protection induced by Toxoplasma gondii virus-like particle vaccine upon intraperitoneal route challenge Dong-Hun Lee a , Ah-Ra Kim a , Su-Hwa Lee a , Fu-Shi Quan b,∗ a b

Department of Biomedical Science, Graduate School, Kyung Hee University, Seoul, Republic of Korea Department of Medical Zoology, Kyung Hee University School of Medicine, Seoul, Republic of Korea

a r t i c l e

i n f o

Article history: Received 23 March 2016 Received in revised form 29 July 2016 Accepted 16 August 2016 Available online 30 August 2016 Keywords: Toxoplasma gondii Virus-like particle Inner membrane complex Vaccine Protection

a b s t r a c t The inner membrane complex sub-compartment has a critical role in Toxoplasma gondii endodyogeny. In this study, we investigated the protection upon intraperitoneal route (IP) challenge induced by the virus-like particles (VLPs) vaccine containing Toxoplasma gondii IMC ISP (RH strain) (Type I). Intranasal immunization with the VLPs in mice elicited enhanced systemic and mucosal Toxoplasma gondii-specific IgG, IgG1, IgG2a and IgA antibody responses, and CD4+ and CD8+ responses. Immunized mice significantly reduced T. gondii cyst burden and size in brain, resulting in cross-protection upon T. gondii (ME49) (Type II) challenge infection. These results indicate that the IP route challenge infection induced by T. gondii IMC ISP VLPs might be a very good target for vaccination representing novel approach to reduce infection. © 2016 Published by Elsevier B.V.

1. Introduction T. gondii infections occur throughout the world, and infection rates differ significantly by country (Pappas et al., 2009). Humans and other warm-blooded animals are its hosts (Dubey, 2004). Approximately one-third of all humans have been exposed to this parasite. Although usually asymptomatic in immunocompetent adults, it can cause severe disease manifestations and even death in immunocompromised subjects. Effective drug are not widely available, and there is no licensed vaccine. Currently, the strategy of toxoplasmosis control is the chemotherapy targeting the acute phase of infection. However, the drugs have toxic effects and do not eliminate the cysts, therefore the patient can have reactivations (Rodriguez and Szajnman, 2012; Bhopale, 2003; Hassan et al., 2014). Regardless of the vaccine construct, the vaccines have not been able to induce protective immunity when the organism is challenged with T. gondii, either directly or via a vector (Jongert et al., 2009). Toxoplasma gondii possess an unusual double membrane structure located directly below the plasma membrane named the inner membrane complex (IMC) coupled to a supporting cytoskeletal network (Harding and Meissner, 2014). IMC sub-compartment proteins (ISPs) have recently been shown to play a role in

∗ Corresponding author. E-mail address: [email protected] (F.-S. Quan). http://dx.doi.org/10.1016/j.actatropica.2016.08.025 0001-706X/© 2016 Published by Elsevier B.V.

asexual T. gondii daughter cell formation. Three proteins, IMC sub-compartment protein (ISP) 1, ISP2, and ISP3, were initially identified and found to localize to distinct sub-compartments of the IMC in T. gondii (Beck et al., 2010). These three proteins localize in the different regions of complex. Disruption of ISP2 caused a significant loss in parasite fitness and a severe defect in endodyogeny, the form of internal cell budding in which two daughter cells are formed within the intact mother parasite (Beck et al., 2010). Since these three ISP1, 2 and 3 are largely conserved in amino acid sequences, we hypothesized that humoral or cellular immunityinducing VLPs containing IMC ISP3 could target all three IMC ISP. Virus-like particle (VLP) vaccines are genetically engineered complexes of multiple copies of protein antigens in a particulate virus-like structure. Viral proteins presented as VLPs or recombinant vaccines are highly immunogenic and induce protection (Zhan et al., 2007; Murawski et al., 2010; Takimoto et al., 2004; Yu et al., 2008). Therefore, it is hypothesized that VLPs containing Toxoplasma gondii IMC will induce strong Toxoplasma gondii-specific immune responses and immunity. VLPs consisting of the respiratory syncytial virus (RSV) fusion F protein and attachment G glycoprotein together with influenza M1 protein have been successfully developed, showing spherical particle shapes of VLPs. In this study, we developed VLPs consisting of the influenza M1 protein as a core protein together with IMC of T. gondii. Immune responses and protection against T. gondii (ME49) challenge were determined in mice immunized with these VLPs.

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2. Materials and methods

2.7. Immunization and challenge

2.1. Parasites, cells and antibodies

Balb/c mice, 6–8 week old, (NARA Biotech, Seoul, Korea) were intranasally immunized twice with 100 ␮g total VLP protein at 4week intervals (n = 6 per group). Blood samples were collected by retro-orbital plexus puncture before immunization and at 1, 2 and 4 weeks after priming and boosting. For challenge studies, naïve or immunized mice were infected with T. gondii ME49 intraperitoneally with 20 cysts in 100 ␮l PBS at 1 month after boosting. Body weight changes and survival were observed daily, and cysts in the brain were counted. Two independent experiments were performed. All animal experiments and husbandry involved in the studies presented in this manuscript were conducted under the guidelines of the Kyung Hee University IACUC.

Toxoplasma gondii RH and ME49 strains were maintained by serial intraperitoneal passage (RH) or oral passage (ME49) in Balb/C mice. Spodoptera frugiperda Sf9 cells were maintained in suspension in serum-free SF900 II medium (GIBCO-BRL) at 27 ◦ C in spinner flasks at 70–80 rpm. Horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin A (IgA) and G (IgG), IgG1, and IgG2a were purchased from Southern Biotech (Birmingham, AL, USA). 2.2. Toxoplasma gondii antigen T. gondii RH tachyzoites were harvested from the peritoneal cavity of the mice 4 days after infection. Cellular debris was removed and the parasites were sonicated, and T. gondii antigen was prepared as described (Fang et al., 2010). 2.3. Constructions of rBV expressing Toxoplasma gondii IMC and influenza M1 Total RNA of T. gondii (RH) tachyzoites was extracted (RNeasy Mini kit; Qiagen, Valencia, CA, USA). Complementary DNA (cDNA) was synthesized and Toxoplasma gondii IMC gene was amplified by polymerase chain reaction (PCR) from cDNA with primers 5-AAAGAATTCACCATGGGAGCTGTCAGCTCG-3 and 5-TTACTCGAGCTATGCCTTCAGCTTCAA-3 (EcoRI and XhoI underlined). A cDNA fragment containing the gene was cloned into pFastBac vector (Invitrogen, Carlsbad, CA, USA) as described previously (Quan et al., 2010). The accession number of the IMC protein in NCBI is HQ012578. Influenza M1 gene was obtained as described (Quan et al., 2010). 2.4. Generation of recombinant baculovirus (rBV) Transfection of DNA containing T. gondii IMC or influenza M1 was done using Cellfectin II (Invitrogen) with SF9 cells as recommended by the manufacturer, followed by transformation of pFastBac containing T. gondii IMC or M1 with white/blue screening. The rBVs were derived using a Bac-to-Bac expression system (Invitrogen) according to the manufacturer’s instructions.

2.8. Antibody responses in sera, feces and intestines Blood and feces samples were collected at weeks 1 and 4 before and after challenge infection. Intestine samples were collected at week 4 postchallenge. IgG, IgG1, IgG2a and IgA antibody responses were determined by ELISA. Plates were coated with 100 ␮l of T. gondii RH (4 ␮g/ml) per well in coating buffer at 4 ◦ C overnight. Feces samples were incubated in PBS at 37 ◦ C for 1 h and the supernatants were collected after centrifugation at 2000 rpm and stored −20 ◦ C until use. Intestines were collected at 4 weeks after postchallenge infection. The small intestine site for each mouse was 10 cm beneath the stomach. The collected intestine was incubated in PBS at 37 ◦ C for 1 h. The intestinal mucus was collected and centrifuged at 2000 rpm for 10 min as described previously (Chu et al., 2014). The supernatant was stored at −20 ◦ C until use.

2.9. Antibody-secreting cell response Spleen was used to detect antibody-producing cells (ASC). T. gondii (RH) (2 ␮g/ml in 100 ␮l) was used to coat 96-well culture plates (SPL), and freshly isolated cells from the spleen (1 × 106 cells/well) were added to each well and incubated for 3–4 days at 37 ◦ C with 5% CO2 . Parasite-specific IgG, IgG1, IgG2a and IgA antibodies secreted into the culture medium and bound to the coated antigens were determined as previously described (Chu et al., 2014).

2.10. Cellular responses 2.5. Production of VLPs Sf9 insect cells were co-infected with recombinant rBVs expressing T. gondii IMC or M1. VLPs released into the cell culture supernatants were harvested and purified through a 15%-30%-60% discontinuous sucrose gradient at 28000 rpm for 1 h at 4 ◦ C. VLP bands between 30% and 60% were collected and then diluted with PBS and pelleted at 28000 rpm for 1 h at 4 ◦ C. VLPs were resuspended in PBS overnight at 4 ◦ C. 2.6. Characterization of VLPs VLPs containing T. gondii IMC and influenza M1 were characterized by Western blots and electron microscopy. Antibody to determine T. gondii IMC in VLPs was prepared from T. gondii ME 49 infected mice. Anti-M1 antibody was used to determine M1 protein content. Negative staining of VLPs was performed followed by transmission electron microscopy, which was done at the Korea Advanced Institute of Science and Technology.

Mouse spleen was collected 1 month after challenge, and singlecell suspensions were prepared from each spleen. Cells were cultured and stimulated with 100 ␮l of 0 or 2 ␮g/ml T. gondii RH. Supernatants of spleen cell cultures were used to determine cytokines interferon-gamma (IFN-␥), interleukin (IL)-6 and IL-10 using OptEIA sets (BD Bioscience, San Jose, CA, USA). To determine CD4+ and CD8+ T cells in the spleen, cells were stained with CD4 and CD8 markers (BD Biosciences) and analyzed using a FACScan flow cytometer (BD, Mountain View, CA, USA). Results were analyzed using WinMDI 2.9 software (De Novo Software, Los Angeles, CA, USA).

2.11. Statistics All parameters were recorded for individuals within all groups. Statistical comparisons of data were carried out using the t-test of the SigmaPlot (Systat Software, La Jolla, CA, USA). A P value < 0.05 was considered to be significant.

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Fig. 1. Characterization of virus-like particles (VLPs). A: Western blot analysis. VLPs (20, 10, 5 ␮g) were loaded for SDS-PAGE. Polyclonal mouse anti-T. gondii antibody was used to probe T. gondii IMC protein and anti-M1 monoclonal antibody was used to determine influenza M1 protein. B: Electron microscopy and VLP size determination. Negative staining of VLPs was performed followed by transmission electron microscopy.

Fig. 2. Toxoplasma gondii-specific antibody responses in sera on immunization. Mice were immunized twice with VLPs as indicated with a 4-week interval. T. gondii-specific IgG, IgG1 and IgG2a antibody responses in the sera were determined after prime and boost (A, B and C). imm: immunization.

3. Results 3.1. Generation of VLPs VLPs were generated by co-infections of rBVs expressing T. gondii IMC or influenza M1. VLPs incorporated with T. gondii IMC and M1 into VLPs were confirmed by Western blot using anti T. gondii polyclonal antibody and M1 monoclonal antibody (Fig. 1A). T. gondii IMC VLPs showed spherical shapes with spikes on their surfaces (Fig. 1B). 3.2. Antibody responses in vaccinated mice before challenge infection The levels of T. gondii-specific IgG, IgG1, IgG2a in sera and IgA and IgG antibody responses in feces after priming and boosting were determined. Total IgG and IgG1, and IgG2a responses from the mice immunized with VLPs showed significantly higher titers after boost compared with those after prime at week 1 and 4, indicating the progressive maturation of T. gondii IMC-specific antibody (Fig. 2A–C). Also, higher levels of IgA and IgG responses in feces were observed after boost compared to naïve control (Fig. 3A and B), indicating mucosal immunity was elicited. These results indicate that VLPs containing T. gondii IMC are highly immunogenic against T. gondii, resulting in higher levels of systemic and mucosal antibody responses. 3.3. Toxoplasma gondii-specific antibodies in mice upon challenge infection T. gondii-specific antibody response profiles in serum, feces and intestines upon challenge infections were determined. Significantly higher levels of IgG, IgG1 and IgG2a antibody responses reactive to T. gondii antigen in sera were detected at week 4 upon challenge (*P < 0.05, **P < 0.01; Fig. 4A–C). T. gondii-specific IgA and IgG antibody

responses in mucosal sites in feces and intestines were also measured. Higher levels of T. gondii-specific IgA and IgG antibodies were detected in feces (Fig. 4D and E; *P < 0.05, **P < 0.01) and intestines (Fig. 4F and G; *P < 0.05, **P < 0.01). These results indicate that IgA and IgG antibodies were rapidly boosted by subsequent infection with T. gondii. Vaccinated mice displayed higher levels of systemic and mucosal antibody responses upon challenge infections. 3.4. VLPs vaccination induced protection upon Toxoplasma gondii challenge infection To assess VLP vaccine efficacy, cyst load and cyst size in brain following intraperitoneal challenge infection were determined as described in the method section. As shown in Fig. 5A, B, significantly decreased cyst counts and cyst sizes in brain were detected in mice upon challenge infections compared to non-immunized mouse controls (Reduction rate of cyst count: 75%, Fig. 5A, **P < 0.01; Reduction rate of cyst size: 50%, Fig. 5B, *P < 0.05), indicating that protective efficacy was induced after challenge infection. Body weight changes were also determined after challenge infections. As shown in Fig. 5C, immunized mice gained body weight whereas control mice lost body weight or died upon challenge. All mice immunized survived whereas control mice showed 60% survival (Fig. 5D). The results indicate that VLP immunized mice showed significantly decreased cyst counts and cyst sizes, indicating protective immunity was induced against Toxoplasma gondii infection. 3.5. T. gondii-specific antibody-secreting cell response Antibody-secreting cell responses were determined at week 4 after challenge infection with T. gondii. We found that significantly higher levels of IgG, IgG1 and IgG2a antibodies specific to T. gondii were secreted into culture supernatants by spleen cells of immunized mice than from cells derived from mice infected only with T. gondii (Fig. 6A–C, *P < 0.05, **P < 0.01). We also evaluated the IgA

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Fig. 3. T. gondii-specific IgG and IgA antibody responses in the feces on immunization. Mice were immunized twice with VLPs as indicated with a 4-week interval. T. gondii-specific IgG and IgA antibody responses in the feces were determined after prime and boost (A and B). immu: immunization.

Fig. 4. Toxoplasma gondii-specific antibodies responses upon challenge infection. Immunized mice were challenge infected orally with T. gondii ME49 at week 4 after boost and T. gondii-specific IgG (A), IgG1 (B) and IgG2a (C) antibody responses in the sera were determined (A, B and C; *P < 0.05, **P < 0.01). IgA and IgG antibody responses from feces and intestine were also determined at week 4 postchallenge (D-G; *P < 0.05, **P < 0.01). Cha: challenge.

antibodies in the same culture supernatants. Significant levels of IgA antibodies specific to T. gondii were detected after 4 days of culture (Fig. 6D, *P < 0.05, **P < 0.01). Taken together, these results indicate that generate memory B cells that have the capacity to rapidly differentiate into antibody-secreting cells upon infection with T. gondii.

3.6. Cytokine production and T cell responses Splenocytes were harvested 1 month after challenge infection, and the levels of production of IFN-gamma, IL-6 and IL-10 from cytokine-secreting cells were determined. Significantly higher levels of IFN-gamma, IL-6 and IL-10 were produced following T. gondii

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Fig. 5. Cyst counts and cyst sizes in brain and body weight changes upon challenge infection. Immunized mice were challenge infected intraperitoneally with T. gondii (ME49) at week 4 after boost. The experiment was repeated twice. At week 4 after challenge infections, mice were sacrificed and cyst counts (A) and cyst sizes (B) in brain were determined (*P < 0.05, **P < 0.01). Body weight changes and survival were also determined daily upon challenge infection (C and D).

Fig. 6. Antibody secreting cell responses. Immunized mice were challenge infected with T. gondii (ME 49) at week 4 after boost and antibody-secreting cells (ASC) in the spleen were determined at week 4 after challenge infections. IgG, IgG1, IgG2a and IgA-secreting cell responses were seen as indicated (A–D; *P < 0.05, **P < 0.01).

antigen stimulation compared to controls (Naïve or Naïve + Cha) (Fig. 7A–C, *P < 0.05, **P < 0.01), indicating that immunized mice induced higher levels of mixed Th1/Th2 cytokine responses upon challenge infections. As shown in Fig. 7D, E, both CD4+ T cells and CD8+ T cells were raised from immunized mice upon challenge infections compared to non-immunized control mice (Fig. 7D, E, *P < 0.05).

4. Discussion The virus-like particles (VLPs) we produced targeted T. gondii IMC ISP, which has critical roles in parasite replication and invasion. Disruption of proteins in IMC produces a severe defect in the form of internal cell budding of T. gondii (Beck et al., 2010; Ouologuem and Roos, 2014). Respiratory syncytial virus VLPs containing influenza

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Fig. 7. T cell responses. Immunized mice were challenge infected and at week 4 after challenge infections mice were sacrificed. IFN-␥, IL-4 and IL-6 cytokines (A, B and C; *P < 0.05, **P < 0.01)), and CD4+ T cell and CD8+ T cell populations in the spleen were determined (D and E; *P < 0.05).

M1 core protein have been demonstrated to induce protective vaccine efficacy, indicating pseudotyped VLPs could generate spherical membrane-enclosed particles and provide protection (Quan et al., 2011). We hypothesized that immunity induced by a T. gondii VLP vaccine containing IMC ISP of T. gondii could prohibit internal cell budding of parasite (endodyogeny), so that, vaccine could protect host from challenge infection. We found that T. gondii VLPs generated are morphologically spherical, and pseudotyped with T. gondii IMC ISP. VLP vaccine elicited significantly higher levels of T. gondii-specific IgG, IgG1 and IgG2a antibody responses than non-immunized control mice. VLP vaccine significantly reduced cyst replication in the brain upon challenge. Thus, T. gondii IMC ISP was proved to be immunogenic in VLPs for the first time. VLP vaccination elicited IgG2a dominant T. gondii-specific IgG antibody responses upon challenge infection which might contribute to significantly reduced cyst counts and cyst size in the brain. This is encouraging since the T. gondii IMC ISP protein might be a very good target for vaccination representing novel approach to reduce infection. The strains of T. gondii used for challenge infection and the strain of mouse and route of infection are critical for the outcome of infection (Johnson, 1984; Blackwell et al., 1993). The susceptibility or resistance of different mouse strains to the infection of ME49 strain is different, where BALB/c mice are susceptible by intraperitoneal (IP) route infection (Subauste, 2012). Challenge infection with intraperitoneal route has been demonstrated to induce better protection than oral route infection in T. gondii vaccine study (Zorgi et al., 2011). T. gondii was initially divided into three genotypes, Type I, II, and III (Howe and Sibley, 1995). Strains of T. gondii differ in their virulence for mice. The RH strain (Type I) is uniformly virulent (lethal) for naive mice. ME49 strain (Type II) is considered relatively avirulent, since they may or may not cause death of naive mice (Subauste, 2012). C56 type III strain is rarely associated with diseases (Yan et al., 2012). Recent study revealed that globally diverse T. gondii isolates comprise 6 major clades, showing biphasic pattern (Su et al., 2012; Pfaff et al., 2014). In our study, T.

gondii (RH) type I was used to clone T. gondii IMC ISP gene for the VLPs generation, and T. gondii (ME49) type II was used for IP route challenge infection in highly susceptible BALB/c mice. Compared to that 50% of non-immunized naïve mice died, all immunized mice were survived (100%), gained weight and reduced 75% of cysts of T. gondii in brain, resulting in promising vaccine efficacy. The results indicated that cross-protective immunity induced by VLPs containing IMC ISP type I T. gondii is working against type II T. gondii strain that is the most prevalent in Europe, especially in immunocompromised patient. We could conclude that our T. gondii VLPs containing IMC ISP is an attractive vaccine candidate. Protective immunity has been evaluated in mice immunized with recombinant proteins, such as surface protein SAGs, or cell invasion-related ROPs, AMAs, MICs and GRAS (Letscher-Bru et al., 2003; Dlugonska, 2008). Mice immunized with recombinant proteins showed little protection against challenge infection with no reduction of cysts in brain. However, VLPs carrying selected T and B cell epitopes from the P. falciparum and P. vivax CS proteins can elicit sterile immunity against blood stage malaria (Whitacre et al., 2015). VLP-peptide display can identify immunogenic mimics of a complex conformational epitope on the plasmodium falciparum blood stage antigen (Crossey et al., 2015). In our current study, mice intranasally immunized with viruslike particles containing IMC ISP exhibited greater protection upon genetically distinct strain T. gondii (ME49). Importantly, mucosal immune responses induced by intranasal vaccination might offer a broader protection against antigenically distinct strains (Quan et al., 2012). VLP vaccination elicited humoral and mucosal immunity through the detection of antigen specific IgG and IgA in serum or in feces. Our analysis of IgG, IgG1, IgG2a and IgA antibodies showed an interesting finding that antibody responses were raised significantly upon challenge infections, indicating greater boosting effect of vaccine. The higher levels of IgG2a than that of IgG1 after immunization and challenge infection were observed, indicating that Th1-type immune responses are dominant. Consistence with this, in current study, higher level of Th1 cytokine IFN-␥ was detected

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than that of Th2 cytokines IL-6 and IL-10. An important goal for vaccination is to induce long-lived memory B cell responses. The present study demonstrated that immunization with VLPs can induce B cells that can rapidly differentiate into antibody secreting plasma cells (ASC) upon exposure to antigens. In our study, VLP vaccination showed increased the activation of CD4+ and CD8+ T cells in the spleen, which might contribute to CD8+ T cell mediated cytotoxicity against cells infected with T. gondii challenge infections (Montoya et al., 1996). Conflicts of interest statement The authors have no conflicts of interest to declare. Acknowledgments This work was supported by a grant from the National Research Foundation of Korea (NRF) (NRF-2014R1A2A2A01004899), and a grant from the Ministry of Health & Welfare, Republic of Korea (HI15C2928). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actatropica.2016. 08.025. References Beck, J.R., Rodriguez-Fernandez, I.A., de Leon, J.C., Huynh, M.H., Carruthers, V.B., Morrissette, N.S., Bradley, P.J., 2010. A novel family of Toxoplasma IMC proteins displays a hierarchical organization and functions in coordinating parasite division. PLoS Pathog. 6, e1001094. Bhopale, G.M., 2003. Development of a vaccine for toxoplasmosis: current status. Microb. Infect 5, 457–462. Blackwell, J.M., Roberts, C.W., Alexander, J., 1993. Influence of genes within the MHC on mortality and brain cyst development in mice infected with Toxoplasma gondii: kinetics of immune regulation in BALB H-2 congenic mice. Parasite Immunol. 15, 317–324. Chu, K., Kim, S., Lee, S., Lee, H., Joo, K., Lee, J., Lee, Y., Zheng, S., Quan, F., 2014. Enhanced protection against Clonorchis sinensis induced by co-infection with Trichinella spiralis in rats. Parasite Immunol. 36, 522–530. Crossey, E., Frietze, K., Narum, D.L., Peabody, D.S., Chackerian, B., 2015. Identification of an immunogenic mimic of a conserved epitope on the plasmodium falciparum blood stage antigen AMA1 using virus-Like particle (VLP) peptide display. PLoS One 10, e0132560. Dlugonska, H., 2008. Toxoplasma rhoptries: unique secretory organelles and source of promising vaccine proteins for immunoprevention of toxoplasmosis. J. Biomed. Biotechnol. 2008, 632424. Dubey, J., 2004. Toxoplasmosis?a waterborne zoonosis. Vet. Parasitol. 126, 57–72. Fang, R., Feng, H., Nie, H., Wang, L., Tu, P., Song, Q., Zhou, Y., Zhao, J., 2010. Construction and immunogenicity of pseudotype baculovirus expressing Toxoplasma gondii SAG1 protein in BALB/c mice model. Vaccine 28, 1803–1807. Harding, C.R., Meissner, M., 2014. The inner membrane complex through development of Toxoplasma gondii and Plasmodium. Cell. Microbiol 16, 632–641. Hassan, I.A., Wang, S., Xu, L., Yan, R., Song, X., Li, X., 2014. DNA vaccination with a gene encoding Toxoplasma gondii Deoxyribose Phosphate Aldolase (TgDPA) induces partial protective immunity against lethal challenge in mice. Parasites Vectors 7, 7–431 (431–3305-7-431).

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