Ascariasis remains the most common helminth infection in humans. As an alternative or complementary approach to global deworming, a pan-anthelminthic vaccine is under development targeting Ascaris, hookworm, and Trichuris infections. As16 and As14 have previously been described as two genetically related proteins from Ascaris suum that induced protective immunity in mice when formulated with cholera toxin B subunit (CTB) as an adjuvant, but the exact protective mechanism was not well understood. As16 and As14 were highly expressed as soluble recombinant proteins (rAs16 and rAs14) in Pichia pastoris. The yeast-expressed rAs16 was highly recognized by immune sera from mice infected with A. suum eggs and elicited 99. 6% protection against A. suum re-infection. Mice immunized with rAs16 formulated with ISA720 displayed significant larva reduction (36. 7%) and stunted larval development against A. suum eggs challenge. The protective immunity was associated with a predominant Th2-type response characterized by high titers of serological IgG1 (IgG1/IgG2a > 2000) and high levels of IL-4 and IL-5 produced by restimulated splenocytes. A similar level of protection was observed in mice immunized with rAs16 formulated with alum (Alhydrogel), known to induce mainly a Th2-type immune response, whereas mice immunized with rAs16 formulated with MPLA or AddaVax, both known to induce a Th1-type biased response, were not significantly protected against A. suum infection. The rAs14 protein was not recognized by A. suum infected mouse sera and mice immunized with rAs14 formulated with ISA720 did not show significant protection against challenge infection, possibly due to the protein’s inaccessibility to the host immune system or a Th1-type response was induced which would counter a protective Th2-type response. Yeast-expressed rAs16 formulated with ISA720 or alum induced significant protection in mice against A. suum egg challenge that associates with a Th2-skewed immune response, suggesting that rAS16 could be a feasible vaccine candidate against ascariasis.
Ascaris lumbricoides, Trichuris trichiura and the hookworm Necator americanus are the three major soil-transmitted helminths (STH) that infect more than one billion poor people in the world and are the leading neglected tropical diseases (NTDs) in terms of disability-adjusted life years (DALYs) [1]. New estimates from the Global Burden of Disease Study 2015 indicate that approximately 761 million people are chronically infected with A. lumbricoides, resulting in 2,700 annual deaths from ascariasis [2,3]. Global control of STH infections depends on the mass drug administration of anthelminthics such as albendazole or mebendazole targeting children between the ages of 1–14 years [4]. However, the rapid rates of post-treatment re-infection [5], potential drug resistance [6], low treatment coverage for children [7], and low access to clean water [8] compromise the effect of anthelminthics alone as a suitable means to control or eliminate STH infections. Indeed, two systematic reviews have largely failed to confirm the beneficial effects of periodic deworming [9,10]. Thus, the development of a multivalent pan-anthelminthic vaccine targeting all three major STH infections would be a desirable biotechnology to prevent parasite reinfection and advance efforts for the control and elimination of these diseases [11]. To advance such a strategy, two major N. americanus hookworm vaccine antigens are undergoing clinical vaccine tests, but there is a need to simultaneously develop A. lumbricoides and T. trichiura candidate antigens as suitable vaccines to be integrated within the human hookworm vaccine development program [12]. With regard to the development of A. lumbricoides vaccine antigens, the genetically highly homologous pig parasite, Ascaris suum, is commonly used as a model to identify and evaluate vaccine candidates. A. suum and A. lumbricoides are morphologically, immunologically, and genetically very similar [13,14] and might even be subspecies variants. Indeed, A. suum has been shown to be an important cause of human ascariasis [15]. Similar to its natural host, the pig, mice can be infected with A. suum eggs and larvae will be released into the mouse intestine from which they will migrate to the lungs. However, in mice, these larvae cannot return to the intestine to develop into adult Ascaris worms [16–18]. Nonetheless, the mouse model has proven to be a valuable tool in the identification and evaluation of vaccine candidates against Ascaris infections [19–21]. Indeed, mice infected with A. suum eggs produced significant protection against A. suum egg challenge as judged by the significant reduction in the number of larvae migrating to the lungs or livers [19,20,22] and also by reduced lung pathology [23]. Using serum from infected mice or rabbit, several immunodominant antigens have been identified including As16 [20], As14 [19], As24 [24], As37 [21] and As-Enol (enolase) [25], and protective immunity has been induced by immunization with recombinant proteins [19,20,26,27] and with DNA [28]. As14 and As16 were the first two antigens previously identified by the Tsuji lab through immunoscreening of an A. suum cDNA library with serum from Ascaris infected rabbit [19,20]. They share 47% amino acid sequence identity and similar localization (in larva and adult stages, as well as in excretory/excretory products) [11]. Intranasal immunization with Escherichia coli expressed recombinant As16 and As14 conjugated with the cholera toxin B subunit (CTB) produced significant protection against A. suum infective egg challenge in mice [19,20]. In addition, rAs16 induced protection in a pig animal model [29], and mice fed with As16-transgenic rice mixed with CTB were also protected against A. suum infection [30]. As14 fused with CTB was also successfully expressed in transgenic rice, but there was no oral immunization and protection reported [31]. Notably though, without CTB as the adjuvant, neither As16 nor As14 were able to induce protective immunity in any model. Here, we report the production of recombinant As16 and As14 in the yeast Pichia pastoris, a eukaryotic expression system with scalability and without the concern for endotoxin contamination as in E. coli-expressed proteins [32]. Mice immunized with yeast-expressed rAs16 mounted a Th2-biased immune response and showed significant protection in terms of lung larval reduction. However, the same immunization regime for rAs14 did not induce any protection in mice. The immunological mechanisms underlying rAs16-induced protection were evaluated compared to rAs14 that did not induce protection. The results in this study provide a feasible approach to developing a vaccine against ascariasis on the basis of the yeast-expressed rAs16 that can be produced at low cost and formulated with alum, an FDA-approved adjuvant for human use that induces the Th2 immune response necessary to achieve anti-Ascaris immunity.
All animal procedures were conducted in accordance with Baylor College of Medicine Institutional Animal Care and Use Committee (IACUC) approved protocol AN-6297 in compliance with the Animal Welfare Act, PHS Policy, and other Federal statutes and regulations relating to animals and experiments involving animals. A. suum eggs were originally obtained from an adult female worm collected from an infected pig at a pig slaughter house near Belo Horizonte, Brazil, and maintained in 0. 2 N H2SO4 until most of them had developed into the embryonated infective stage (50–250 days). The infective embryonated eggs were shipped to our lab in Houston and used to orally challenge BALB/c mice as previously described [17]. The A. suum larvae hatch in the mouse intestine, and then migrate to the liver and lungs. The number of larvae recovered from mouse lung tissue eight days post infection were used as a biomarker to evaluate vaccine efficacy [17]. Crude extracts of A. suum eggs and lung-stage larvae were prepared by homogenization and sonication, and the insoluble pellet was removed by centrifugation as previously described [33]. Amino acid sequences were aligned using CLUSTAL W and prepared for display using BOXSHADE. The phylogenetic trees were generated for As16 and its homologues from different nematodes using Phylogeny. fr [34] (http: //www. phylogeny. fr/index. cgi). DNA coding for As16 without its signal peptide was codon optimized for expression in yeast and synthesized by GenScript. The DNA coding for As14 without its signal peptide was PCR amplified with As14 specific primers from A. suum larvae cDNA reverse-transcribed from total larval RNA. As16 and As14 coding DNAs were subcloned into the yeast expression vector pPICZαA (ThermoFisher Scientific, Carlsbad). The correct sequences and reading frames of the recombinant plasmids were confirmed by double-stranded DNA sequencing using vector flanking primers, α-factor and 3’-AOX1. The recombinant As16 and As14 (rAs16 and rAs14) with a hexahistidine tag at its C-terminus were expressed in yeast stain P. pastoris X-33 under induction with 0. 5% methanol for 48–72 hours and then purified by immobilized metal ion affinity chromatography (IMAC), as described previously [35]. The purity of the recombinant proteins was determined by SDS–PAGE. The protein concentration was measured using BCA (ThermoFisher Scientific, Waltham) and Endotoxin clearance was confirmed using the Charles River Endosafe-PTS system (Charles River, Houston). Six-week old female BALB/c mice were purchased from Taconic and divided into four groups of 20 animals each. Two vaccine groups were immunized subcutaneously with 50 μg of rAs16 or rAs14 emulsified with the adjuvant Montanide ISA720 (Seppic, Paris, France) in a total volume of 100 μl (antigen/ISA720 = 30/70 v/v). Mice were boosted twice with the same dose on days 21 and 35. The control groups were injected with PBS or PBS+ISA720 using the same regimen. Two weeks after the final vaccination, 5 mice from each group were sacrificed and blood and splenocytes were harvested for immunological tests. The remaining 15 mice from each group were challenged with 2,500 A. suum embryonated eggs in a total volume of 100 μl, administered by oral gavage. Eight days after infection, all infected mice were sacrificed, lungs were harvested, and A. suum lung-stage larvae were collected using a Baermann apparatus, as previously described [17]. Reduction in larval burden was calculated in all groups and the results were compared between the vaccine groups and the PBS and adjuvant control groups. To improve the protection induced by rAs16 and interpret the immunological mechanism underlying the As16-induced protective immunity, another vaccine trial was performed by formulating 25 μg of rAs16 with either 200 μg of Alhydrogel (Brenntag, Mülheim, Germany), 20 μg of MPLA (InvivoGen, San Diego), or 50 μl of AddaVax (50/50, v/v) (InvivoGen, San Diego), each administered subcutaneously in a total volume of 100 μl per mouse given using the immunization regimen described above. Control groups were given adjuvant only. As a positive control, one group of 20 mice was orally infected three times with 1,000 A. suum embryonated eggs. After these immunizations, all mice were challenged with 2,500 A. suum embryonated eggs. Sera from all blood samples were isolated and frozen at -20°C. The sera samples were assayed for antigen-specific IgG isotypes (IgG1, IgG2a) by a modified indirect enzyme-linked immunosorbent assay (ELISA). Briefly, individual wells of Nunc-Immuno Maxisorp plates (Thermo Scientific, Waltham) were each coated with 100 μl of rAs16 (3. 1 μg/ml) or rAs14 (0. 39 μg/ml) in coating buffer (KPL, Milford) overnight at 4°C based on the pretested optimal signal/noise ratio. The coated plates were blocked overnight with 0. 1% BSA in PBST (PBS +0. 05% Tween-20), then incubated with diluted serum samples, starting at 1: 200 in 0. 1% BSA in PBST for 2 hours. Horseradish peroxidase (HRP) -conjugated goat anti-mouse IgG1 and IgG2a (Lifespan Biosciences, Seattle) were used as secondary antibodies (1: 4,000 in PBST). Sure Blue TMB (KPL, Milford) was added as the substrate. The reaction was stopped by adding 100 μL of 1 M HCl. The absorbance was measured at 450 nm using a spectrophotometer (BioTek, Winooski). Samples including crude extracts of A. suum lung-stage larvae and eggs and the recombinant proteins were separated by SDS–PAGE, then transferred onto PVDF membrane (ThermoFisher, Waltham). After blocking with 5% (w/v) skim milk powder in PBST, the membrane was incubated with sera from mouse immunized with recombinant proteins or A. suum eggs. HRP-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad) was used as a secondary antibody. The antibody recognized bands were developed by ECL (GE Healthcare, Chicago). Recombinant Tc24 protein, expressed in yeast [36], was used as a negative control. Spleens were obtained from mice two weeks after the third immunization and the splenocytes were disassociated using a 100 μm cell strainer. The cells were then suspended in complete RMPI medium containing 10% heat inactivated FBS and 1x pen/strep solution. After being centrifuged at 300 x g for 5 min, the cells were resuspended in 2 mL ACK lysis buffer (Thermo Scientific, Waltham) for 5 min. After centrifugation the splenocytes were resuspended in complete RPMI media containing 10% DMSO and stored in liquid nitrogen until use. For the cytokine stimulation assay, splenocytes were thawed in a 37°C water bath and transferred to 5 mL pre-warmed complete RPMI. Cells were washed once to remove residual DMSO. Splenocytes were seeded in a 96-well U-bottom culture plate (Falcon, Corning) at 1x106 cells per well in 250 μl medium and re-stimulated with either 25 μg/mL rAs14 or rAs16 at 37°C, 5% CO2 for 48 hours. Positive controls were stimulated with 20 ng/mL PMA and 1 μg/mL Ionomycin, and unstimulated negative control cultures were performed concurrently. After 48 hours the cells were pelleted by centrifugation at 300 x g for 5 min and the supernatants were collected for measuring cytokine production. The supernatant samples were tested for levels of IL-2, IL-4, IL-5, IL-10, IL-12 (p70), GM-CSF, IFN-γ and TNF-α using a Bio-Plex Pro Mouse Th1/Th2 8-plex kit (Bio-Rad, Hercules). To save material and costs, and to increase the sensitivity of the experiment, the kit was used in combination with DA-Bead plates (Curiox Biosystems, Singapore) as previously described [37]. Samples were run on a Bio-Plex Magpix multiplex reader according to manufacturer' s recommendations (Luminex, Austin). Raw Luminex data were analyzed using the Bio-Plex Manager 6. 0 software and plotted in GraphPad Prism 6. 0. The cutoffs of the cytokine standards were dependent on the lot number of the Bio-Rad kit. To remove individual baseline cytokine values, cytokine values from non-restimulated samples were subtracted from those associated with antigen restimulated samples. Statistical significance of differences between groups was determined using a Mann-Whitney test using Prism 6. In Fig 5c the groups receiving the antigen + adjuvant were compared to the associated adjuvant alone group, the A. suum egg group, and to the PBS group using a Fisher’s LSD test. Data was presented as means ± standard deviation. For the statistical analysis, p < 0. 05 was considered to be statistically significant.
The genes encoding As16 (yeast codon optimized) and As14 (native sequence) without their signal peptides were cloned into the Pichia expression vector pPICZαA. The hexahistidine-tagged rAs16 and rAs14 proteins were expressed in P. pastoris X-33 through induction with 0. 5% methanol over 72 hours and then purified by immobilized metal ion affinity chromatography (IMAC). The purified rAs14 and rAs16 proteins were analyzed by SDS–PAGE (Fig 1A). The apparent molecular weight for both proteins was approximately 15. 0 kDa, which corresponds well to the sizes of the predicted gene products (14. 8 kDa for rAs14 and 15. 4 kDa for rAs16). To determine whether rAs14 or rAs16 were recognized by protective immune sera from mice repeatedly infected with A. suum eggs described below (Protective immunity induced by immunization with rAs16), the infected mouse sera were used for Western blot. We observed that only rAs16 was recognized by the infected mouse sera, while rAs14 was not recognized by the same sera (Fig 1B). Even though the two proteins share 47% identity and 66% similarity in sequence (Fig 1E), there was no obvious immunological cross reaction between them when using rAs16 or rAs14 immunized mouse sera individually (Fig 1C and 1D). The results suggest that As16 antigen was exposed to the immune system during A. suum egg infection and larval migration, whereas As14 antigen may not have been immunologically accessible. As16 is a 16 kDa nematode specific protein found among several different nematode species (Fig 2). It is present in different developmental stages of A. suum, including larvae and adult worms, but its function remains unknown [20]. As16 is highly conserved in Ascaris spp. ; it shares 94% sequence identity with its counterpart in the human parasite A. lumbricoides (Al-Ag2), suggesting the possibility of achieving cross-protection for both Ascaris species if As16 were to be used as a vaccine antigen. Its homologues in filarial worms are ranked among the leading vaccine candidates against human onchocerciasis (Ov-RAL-2) [38] and Brugia malayi filarial infections (Bm-RAL-2) [39]. The As16 homologue from the canine hookworm (Ancylostoma caninum, Ac-16) also protected dogs from blood loss and reduced worm fecundity [40], and the Baylisascaris schroederi homologue, Bs-Ag2, protected giant pandas from infection with that parasite [41]. Immunization with rAs16 and rAs14 formulated with the ISA720 adjuvant elicited significant titers of antigen-specific IgG1 and IgG2a antibodies in mice, with IgG1 as the predominant subclass (Fig 3A). The IgG1/IgG2a ratio after As16 immunization (2662: 1) was more than 100-fold higher than the ratio for As14 (206: 1) (Fig 3A), suggesting that a predominant Th2-type immune response occurs to both antigens, but in particular to rAs16. Mice given PBS+ISA720 did not show any IgG isotype responses specific to rAs16 or rAs14. To evaluate the cytokine profiles induced by immunization with rAs16 and rAs14, mice were sacrificed two weeks after the final immunization and their splenocytes were isolated. Cytokine profiles were determined by measuring levels of IL-2, IL-4, IL-5, IL-10 and IFN-γ in supernatants of splenocytes re-stimulated with 25 μg/ml rAs16 or rAs14 for 48 hours. Signal background in blank media was subtracted from re-stimulated samples. Statistically significantly increased levels of the cytokines IL-2, IL-4, IL-5, IL-10 were detected in the supernatants of re-stimulated splenocytes from mice immunized with rAs16 and rAs14; however, IFN-γ was significantly increased only in mice immunized with rAs14, not in the mice immunized with rAs16 (Fig 3B). Splenocytes from the PBS +ISA720 control group did not show any detectable cytokine expression. All mice were orally challenged with 2,500 A. suum embryonated eggs two weeks after the final immunization. A. suum larvae were collected from the lungs of immunized mice eight days after egg challenge. The lung larva count showed that mice immunized with 50 μg of rAs16 formulated with ISA720 adjuvant showed a 36. 7% larva reduction compared to the adjuvant-only control groups, constituting a statistically significant difference (p<0. 001) (Fig 4A). In addition, the size of the larvae collected from rAs16 immunized mice was much smaller than those collected from adjuvant control mice, suggesting developmental stunting due to the vaccination (Fig 4B). However, mice immunized with rAs14 did not show any protection in terms of reducing the number of larvae found in the lungs, or affecting the size of the larvae. To further understand the immunological mechanism underlying the protective immunity induced by rAs16, and to select an adjuvant that performs best in protecting mice from infection, mice were immunized with only half the amount of As16 (25 μg, compared to 50 μg of rAs16 used for formulation with ISA720), formulated with three different adjuvants (Alhydrogel, MPLA and AddaVax). As a positive control, another group of mice was immunized through three trickle infections with 1,000 A. suum eggs. After three immunizations, all mice were challenged with 2,500 A. suum embryonated eggs. Mice immunized with 25 μg of rAs16 formulated with Alhydrogel, an alum adjuvant inducing a predominant Th2-type response, experienced a 38. 9% lung larval reduction, which is statistically significant compared to the PBS and adjuvant-only control groups. However, rAs16 formulated with MPLA, a TLR4 agonist inducing a Th1/Th2-mixed type immune response, induced only a 26. 1% lung larval reduction that was not statistically different from the control groups. Mice immunized with rAs16 formulated with AddaVax, an oil-in-water based adjuvant similar to the MF59 adjuvant that is licensed for flu vaccines in Europe and known to also induce a Th1/Th2 mixed immune response, were not statistically significantly protected against the A. suum egg challenge. Strikingly, mice immunized with three trickle infections with 1,000 A. suum eggs produced almost sterile immunity (99. 6% lung larval reduction) against A. suum challenge (Fig 5A). Serological antibody measurement revealed that mice immunized with rAs16 formulated with three different adjuvants all produced high titers of IgG1 and IgG2a, with a bias towards IgG1 (IgG1/Ig2a = 120–202) (Fig 5B). Interestingly, mice repeatedly infected with a low-dose of A. suum eggs produced a 99. 6% lung larva reduction and also showed a significant increase of anti-As16 specific IgG1 antibodies, but without any accompanying IgG2a response. This suggests native As16 is released and exposed to the host immune system during repeated low-dose A. suum infections and may be involved in the induction of the observed Th2 protective immunity. Cytokines released by splenocytes upon re-stimulation of rAs16 showed that immunization with rAs16 formulated with Alhydrogel induced the release of IL-5, a major cytokine linked to Th2 responses in mice, and some level of IL-12 and GM-CSF. There were no detectable levels of IL-2, IL-4, IL-10, IFN-γ or TNF-α observed in mice immunized with rAs16 + Alhydrogel. Conversely, mice immunized with rAs16 formulated with MPLA and AddaVax showed a significant release of Th1 associated cytokines (IL-12, IFN-γ and TNF-α), Th2 type cytokines (IL-4, IL-5), and IL-10, IL-2 and GM-CSF as well (Fig 5C), even though there was no significant protective immunity observed in these Th1/Th2 adjuvant groups. Western blot analysis with sera from mice immunized with rAs16 and rAs14 demonstrated that anti-As16 mouse sera recognized a band at ~14 kDa in the soluble extracts of lung larvae of A. suum, but not in the extracts of A. suum eggs. For As14, mouse anti-As14 sera specifically recognized a band of about 13 kDa in lung larval extracts, but not in A. suum eggs, indicating native As16 and As14 are expressed only at the larval stage, not in the eggs of A. suum (Fig 6). As16 and As14 are likely expressed in A. suum larvae after hatching and during the migration to the lungs. However, since infected mice only produced antibodies to As16 but not to As14 (Fig 1), it appears that only As16 is exposed to the immune system during larval migration.
As16 (A. suum, BAC66614. 1); As14 (A. suum, BAB67769. 1); Ce16 (Caenorhabditis elegans, NP_495640. 1); Dv16 (Dictyocaulus viviparus, KJH51207. 1); Acey16 (Ancylostoma ceylanicum, EPB72254. 1); Ac16 (A. caninum, ABD98404. 1); Ad16 (A. duodenale, KIH68079. 1); Sv-SXP (Strongylus vulgaris, AGF90534. 1); Na-SAA2 (Necator americanus, XP_013290850. 1); Al-Ag1 (A. lumbricoides, ACJ03764. 1); Bs-Ag1 (Baylisascaris schroederi, ACJ03761. 1); Av-RAL2 (Acanthocheilonema viteae, AAB53809. 1); Ll-SXP (Loa loa, XP_003142836. 1); Ov-RAL2 (Onchocerca volvulus, P36991. 1); WB14 (Wuchereria bancrofti, AAC17637. 1); Bm-RAL-2 (Brugia malayi, XP_001900036. 1); Bs-Ag2 (B. schroederi, ACJ03762. 1); Al-Ag2 (A. lumbricoides, ADB45852. 1); Hc16 (Haemonchus contortus, CDJ91573. 1); Asim16 (Anisakis simplex, BAF43534), Tc16 (Toxocara canis, KHN84076. 1) and As-RAL-2 (Anisakis simplex, BAF75709. 1).
Roundworms (Ascaris) infect more than 700 million people living in poverty worldwide and cause malnutrition and physical and mental developmental delays in children. As an alternative or complementary approach to global deworming, a pan-anthelminthic vaccine is under development that targets ascariasis in addition to other human intestinal nematode infections. Towards this goal, two Ascaris suum antigens, As16 and As14, were expressed in Pichia pastoris as recombinant proteins. Mice immunized with rAs16 formulated with ISA720 adjuvant produced significant larva reduction (36. 7%) and stunted larval development against A. suum egg challenge. The protection was associated with predominant Th2-type responses characterized by high levels of serological IgG1 (IgG1/IgG2a > 2,000) and Th2 cytokines, IL-4 and IL-5. A similar level of protection was observed in mice immunized with rAs16 formulated with alum that induces mainly a Th2-type immune response, whereas mice immunized with rAs16 formulated with MPLA or AddaVax, both inducing major Th1-type responses, were not significantly protected against A. suum infection. High-yield expression of rAs16 in yeast will allow for large-scale manufacture, and its protective efficacy when formulated with alum suggests its suitability as a vaccine candidate.
innate immune system
medicine and health sciences
immune physiology
cytokines
immunology
parasitic diseases
vaccines
preventive medicine
developmental biology
immunologic adjuvants
molecular development
infectious disease control
vaccination and immunization
public and occupational health
infectious diseases
proteins
life cycles
recombinant proteins
immune response
immune system
biochemistry
helminth infections
immunity
physiology
biology and life sciences
larvae
Yeast-expressed recombinant As16 protects mice against Ascaris suum infection through induction of a Th2-skewed immune response