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Published in final edited form as: Int J Parasitol. 2014 Jun 12;44(10):675–679. doi: 10.1016/j.ijpara.2014.05.003

Vaccination with a genetically modified Brugia malayi cysteine protease inhibitor-2 reduces adult parasite numbers and affects the fertility of female worms following a subcutaneous challenge of Mongolian gerbils (Meriones unguiculatus) with B. malayi infective larvae

Sridhar Arumugam a, Junfei Wei b, Danielle Ward a, David Abraham c, Sara Lustigman d, Bin Zhan b, Thomas R Klei a,*
PMCID: PMC5335902  NIHMSID: NIHMS611062  PMID: 24929131

Abstract

Vaccination of Mongolian gerbils with Brugia malayi cysteine protease inhibitor-2 in which the amino acid Asn66 was mutated to Lys66 (Bm-CPI-2M) resulted in reduced parasite numbers of 48.6% and 48.0 % at 42 and 90 days p.i. with B. malayi L3s. Fertility of female worms was also affected at 90 days p.i. In vitro killing of L3s observed in the presence of gerbil peritoneal exudate cells and anti-Bm-CPI-2M sera suggests antibody-dependent cell-mediated cytotoxicity as a putative protective mechanism. These observations suggest that Bm-CPI-2M is a promising prophylactic and anti-fecundity vaccine candidate.

Keywords: Brugia malayi cysteine protease inhibitor-2 (Bm-CPI-2), Filariasis, Vaccination, Fecundity, Antibody dependent cell-mediated cytotoxicity (ADCC)

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Lymphatic filariasis and onchocerciasis, two major neglected tropical diseases, affect 150 million people worldwide with 1.39 billion people at risk (WHO, 2012). Mass drug administration (MDA) of antifilarial drugs has successfully reduced the transmission of these infectious agents and the disease burden in some endemic areas (WHO, 2012). However, continued MDA can potentially contribute to the development of drug resistance as reported in onchocerciasis (Osei-Atweneboana et al., 2011). Development and distribution of filarial vaccines is another feasible strategy for the global control and elimination of filariasis (Lustigman, 2012; Prichard et al., 2012). The vaccine candidates of Onchocerca volvulus and lymphatic filariae have been reviewed by Lustigman (2012). The level of protection reported ranges from 40% to 76%. Recently, a recombinant fusion protein of Brugia malayi α crystalline domain and C-terminal extension of heat shock protein 12.6 (Bm-HSPαc), B. malayi abundant larval transcript-2 (Bm-ALT-2) and B. malayi tetraspanin large extracellular loop (Bm-TSP LEL) showed more than 95% protection against B. malayi infection in mice (Dakshinamoorthy and Kalyanasundaram, 2013).

In parasitic nematodes, the endogenous native cysteine protease inhibitors (CPIs) or cystatins were shown to be involved in essential developmental processes and in the interactions of the parasite with the vector and the mammalian hosts (de Macedo Soares and de Macedo, 2007; Gregory and Maizels, 2008). In filarial nematodes, CPIs have been characterized in B. malayi, O. volvulus, Litomosoides sigmodontis and Acanthocheilonema viteae (Lustigman et al., 1992; Pfaff et al., 2002; Gregory and Maizels, 2008). Three CPIs, Bm-CPI-1, Bm-CPI-2 and Bm-CPI-3, have been identified in B. malayi. Bm-CPI-2 is secreted by adult female worms and is present in all life stages of the parasite, whereas Bm-CPI-1 and Bm-CPI-3 are found only in the L2 and L3 stages (Manoury et al., 2001; Murray et al., 2005; Gregory and Maizels, 2008). Interestingly, immunization of mice with the O. volvulus recombinant onchocystatin (Ov-CPI-2/Ov-7), when formulated with alum, significantly reduced O. volvulus larval survival by 49% and inhibited development of L3s to L4s in a O. volvulus- diffusion chamber model (Abraham et al, 2001; Lustigman et al., 2002).

In the L. sigmodontis mouse model, DNA vaccination of genetically modified Ls-CPI-2 with Asn66 mutated to Lys66 together with L. sigmodontis abundant larval transcript-1 (Ls-ALT-1) significantly increased their immunogenicity and reduced the number of developing adult worms and microfilaremia (Babayan et al., 2012). Recently, we reported that vaccination of gerbils with recombinant Bm-CPI-1 or -2 did not confer protection against B. malayi infection but rather altered the site of establishment of the developed adult worms, causing an increase in their residence in heart and lungs instead of lymphatic vessels (Arumugam et al., 2014). The reason for the lack of protective efficacy of the native Bm-CPIs vaccine is unclear, but it could be related to the known immunomodulatory property of cystatins, especially Bm-CPI-2. Bm-CPI-2 has an amino acid sequence motif, SND, which constitutes a distinct inhibitory site that is specific for asparaginyl endopeptidase (AEP) in antigen presenting cells (APC) (Manoury et al., 2001). This immunomodulatory property of native Bm-CPI-2 could have evoked a strong immune suppression, dampening the host immune response upon vaccination by inhibiting cysteine proteases of APC.

If the putative immunomodulatory properties of the native Bm-CPI-2 reduced its efficacy as an anti- B. malayi vaccine, it is possible that mutating it, as was done with Ls-CPI-2 (Babayan et al., 2012), would enhance its efficacy as a vaccine. This was tested by vaccinating gerbils with a recombinant protein containing a mutation at amino acid position 66, Asn66 to Lys66 (Bm-CPI-2M). Results indicate that vaccination of gerbils with Bm-CPI-2M reduces B. malayi parasite numbers and affects fertility of female worms. Furthermore, anti-Bm-CPI-2M sera in the presence of gerbil peritoneal exudate cells (PEC) efficiently killed L3s in vitro in an antibody-dependent cell-mediated cytotoxicity (ADCC) manner.

The L3s were harvested from infected mosquitoes, Aedes aegypti, using the previously described technique (Klei et al., 1990). The Institutional Animal Care and Use Committee (IACUC) at Louisiana State University (LSU), USA approved the animal experimental protocols. DNA encoding Bm-CPI-2 (GenBank accession number AF015263), minus the signal peptides, with the immunomodulatory active Asn66 mutated to Lys66 (Bm-CPI-2M) was synthesized by GenScript, USA and recombinant protein was expressed using the pET41a expression vector and the BL21 (DE3) Escherichia coli strain (Novagen, USA) as described previously (Zhan et al., 2002). Gerbils were immunized three times at 2 week intervals. Bm-CPI-2M was formulated with alum (Rehydragel LV, General Chemical, NJ, USA). Performed twice, both experiments consisted of 10, 8 week old male gerbils in each of the two groups; one group was vaccinated with 25 μg of Bm-CPI-2M absorbed to 320 μg of alum in tris buffered saline (TBS) buffer and one group was vaccinated with 320 μg of alum in TBS buffer alone. Sera were collected 1 week prior to the start of each experiment and 1 week following the third immunization. Two weeks after second boost, gerbils were challenged with 100 L3s of B. malayi s.c. in the medial surface of the left leg. In the first and second Bm-CPI-2M vaccination experiments, necropsies were performed 42 and 90 days p.i., respectively. In the first experiment, necropsy at day 42 p.i. was performed to investigate the impact of Bm-CPI-2M vaccination on the development of L3s to adult male and female worms. In the second experiment, necropsy at day 90 p.i. not only allowed for the data of the first experiment to be confirmed but also made it possible to test the effect of the vaccine on the fertility of female worms. Fertility was determined by measuring the levels of microfilariae (MF) in gerbil blood and by performing embryograms of female worms. The total number of worms recovered was recorded, male and female worms were differentiated and tissue location was noted upon harvest from heart, lung and various lymphatic organs as previously described (Arumugam et al., 2014). Female worm length was measured under an Olympus MVX10 microscope using the Olympus DP2-BSW software. Statistical significance was determined by a Mann–Whitney U Test using GraphPad Prism version 4.03. P ≤ 0.05 represents a statistically significant difference between vaccinated and alum adjuvant control groups.

In the first experiment, a significant reduction (48.6%) in worm recovery was observed at 42 days p.i. in the Bm-CPI-2M vaccinated group compared with controls (Fig. 1A). The reduction in worm numbers was seen in both the heart and lung sites and in the lymphatic organs of vaccinated animals (Fig. 1B, C).

Fig. 1.

Fig. 1

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Vaccination with recombinant Brugia malayi Bm-CPI-2M protein confers protection against B malayi L3 infection in Mongolian gerbils. (A) Experiment 1, total number of recovered adult worms (42 days p.i.). (B) Distribution of worms in heart and lungs in experiment 1. (C) Distribution of worms in lymphatic organs in experiment 1. (D) Experiment 2, total number of recovered adult worms (90 days p.i.). (E) Distribution of worms in heart and lungs in experiment 2. (F) Distribution of worms in lymphatic organs in experiment 2. The lines represent the mean with S.Ds.

A significant reduction (48.0%) in worm recovery was also observed in vaccinated animals in the second experiment when necropsies were performed at 90 days p.i. (Fig. 1D). In this experiment, one gerbil in the control group died of unknown causes prior to challenge and thus the group contained only nine animals. As in the first experiment, a reduction in worm numbers was seen in both the heart and lung sites and in the lymphatic organs of vaccinated animals (Fig. 1E, F). However, the results of two experiments differ in that the statistically significant reduction in numbers of worms was seen in the lymphatic organs in the second experiment compared with the larger reduction that was seen in the heart and lungs in animals in the first experiment. The reasons for these statistical differences between tissue sites are unclear but may relate to the timing of the necropsies and subsequent movement of parasites.

We analyzed sex ratios of worms recovered in both of the experiments. While there was a very slight decrease in the female:male ratio in Bm-CPI-2M vaccinated gerbils this was not statistically significant (Supplementary Fig. S1A, B).

Antigen-specific IgG responses were determined by ELISA following the method previously described (Arumugam et al., 2014). In both experiments, a strong antigen-specific IgG response with a final end point titer of up to 1:2,048,000 was seen in sera of vaccinated animals taken post-vaccination prior to L3 challenge. The level of antigen-specific IgG did not correlate with the level of protection seen. The IgG levels were the same between a non-protected vaccinated gerbil with a worm count of 24 compared with a protected vaccinated gerbil with a worm count of 1. The final end point titer was 1: 2,048,000 for both gerbils and there was no antigen-specific IgG in the alum control or pre-immune gerbil serum (Supplementary Fig. S2).

The number of circulating MF was measured in the second experiment at 90 days p.i. in 20 μl of blood from control and vaccinated gerbils at the time of necropsy. Only three gerbils were microfilareamic at 90 day p.i. Two of these were in the control group and one in the vaccinated group. There was no statistical difference in MF levels seen (Supplementary Fig. S3). To further investigate the impact of Bm-CPI-2M vaccination on fertility of female worms, embryograms were performed to enumerate the progeny in individual female worms harvested from control and vaccinated gerbils at 90 days p.i. The worms used for these counts were previously fixed in 70% ethanol containing 30% glycerin. This fixation likely accounts for the slight distortions seen in the recovered developmental stages (Fig. 2A). Nonetheless, differentiation and counting of stages was easily accomplished. The morphology of eggs, early embryos, late embryos, pretzel stage MF and stretched MF are shown in Fig. 2A. Single female worms were transferred to a 1.5 ml tube containing 200 μl of PBS. A disposable pestle was used to gently crush the worms and 300 μl of PBS were used to wash the worm debris from the pestle into the same tube. The tube was centrifuged at 400 g for 2 min and 450 μl of PBS was carefully removed without disturbing the pellet. The pellet with worm content in the remaining 50 μl was mixed well and 10 μl was added to the hemocytometer. The total number of eggs, embryos, pretzel stage MF and stretched MF (Ziewer et al., 2012) was counted. Early and late stage embryos were grouped together as embryos for the purposes of counting. At least two female worms per gerbil were analyzed from each group except for one gerbil in the vaccinated group from which only one female worm was harvested. Embryogram data demonstrated that there was no reduction in the number of eggs/female worm between control and vaccinated gerbils (Fig. 2B). In contrast, there was a significant reduction in the number of embryos, pretzel stage MF and stretched MF per female worm harvested from vaccinated gerbils. (Fig. 2C–E).

Fig. 2.

Fig. 2

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Embryogram data of female Brugia malayi worms harvested from alum only and Bm-CPI-2M vaccinated gerbils. (A) Embryogram picture of female B. malayi showing different developmental stages including eggs, early embryos, late embryos, pretzel stage microfilariae (MF) and stretched MF (40× magnification). (B) Eggs/female worm. (C) Embryos/female worm. D) Pretzel stage MF/female worm. (E) Stretched MF/female worm. The lines represent the mean with S.Ds.

Parasite naïve male gerbils were injected with 3 ml of 3% (w/v) of thioglycolate i.p. to recruit PECs used in in vitro killing assays. Five days after injection, PECs were harvested from the peritoneal cavity and suspended in RPMI supplemented with 10% FCS and penicillin-streptomycin (Sigma, USA). The serum used in the assays was from Bm-CPI-2M immunized gerbils with an end point titer of anti-Bm-CPI-2M specific IgG antibodies of 1:2,048,000. A control serum was from gerbils vaccinated with alum alone. In vitro killing assays were performed twice. Assays were performed in 24-well culture plates containing 1 ml of RPMI medium and 10 L3s were added per well (Chandrasekhar et al., 1985). An initial dilution assay was performed in replicates in the presence of PECs using serum dilutions of 1:20, 1:40, 1:80 and 1:100 from gerbils vaccinated with alum only, anti-Bm-CPI-1, anti-Bm-CPI-2 or anti-Bm-CPI-2M sera. Sera used were recovered following the second vaccination boost. The plate was incubated for 48 h at 37°C with 5% CO2. After 48 h, the survival of L3s was recorded based on their motility and the integrity of their internal tissues. Those that were completely immotile with granulated, irregular tissues within the body were considered dead.

No L3 killing was observed in the presence of 2 × 105 PEC in any of the dilutions of control sera. However, L3 killing of more than 50% was observed in the 1:20 dilution of anti-Bm-CPI-2M sera. This decreased to 15% in the 1:40 dilution and was not seen at higher dilutions. In vitro killing assays with sera from gerbils immunized with the native Bm-CPI-1 or- 2 at 1:20 dilution in the presence of PECs induced only 0–20% L3 killing and no killing was observed above 1:40 dilution of native Bm-CPI-1 or- 2 sera (Supplementary Table S1). No killing was observed in RPMI media controls or when L3s were cultured either in the presence of PEC alone or 1:20 dilutions of alum/Bm-CPI-1 or- 2/Bm-CPI-2M serum alone (Supplementary Table S1).

The initial killing assay performed with different serum dilutions determined that 1:20 dilution of anti-Bm-CPI-2M serum in the presence of 2 × 105 PECs was able to kill more than 50% of cultured B. malayi L3s. The L3 killing assay was repeated with 1:20 anti-Bm-CPI-2M compared with sera from control gerbils vaccinated with alum. Ten L3s were placed in each well corresponding to the following five treatment groups: i) RPMI media; ii) 2 × 105 PECs; iii) 1:20 dilution of anti-Bm-CPI-2M sera; iv) 2 × 105 PECs plus 1:20 dilution of control sera; and v) 2 × 105 PECs plus 1:20 dilution of anti-Bm-CPI-2M sera. In groups i to iv, 100% of cultured L3s survived (Supplementary Movie S1, living L3 cultured with PECs plus control serum), while 60% of L3s were killed in group v, when they were cultured with PECs and anti-Bm-CPI-2M antibodies (Supplementary Video S2, Supplementary Movie S3 - higher magnification). Notably, the remaining L3s incubated with PECs plus anti-Bm-CPI-2M sera were counted as living but were sluggish and contained granules within their bodies (Supplementary Movie S4). Adherence of PECs to the surface of L3s was observed on the dead or dying larvae (Fig. 3B, Supplementary Movies S2–S4), whereas no adherence of PECs was observed in L3s incubated with PECs plus sera from the alum control group (Fig. 3A, Supplementary Movie S1).

Fig. 3.

Fig. 3

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Images of Brugia malayi L3s in in vitro killing assays. (A) L3s cultured in the presence of peritoneal exudate cells (PECs) plus 1:20 dilution of gerbil sera from the alum control group. (B) L3s cultured in the presence of PECs plus 1:20 dilution of gerbil sera from the Bm-CPI-2M immunized gerbils. Arrows indicate adherence of cells to dead L3s (40× magnification).

Adherence of PECs to the surface of L3s followed by killing of L3s in the presence of anti-Bm-CPI-2M sera suggests that ADCC is a potential mechanism of immune response induced by the vaccination that resulted in a reduction in the number of adult worms.

We did not detect any evidence of adult killing at the time of necropsy. There was no attachment of cells or formation of granulomas around the adult worms in any group and all recovered worms were alive. There was no significant difference in the length of female worms recovered from alum control groups and Bm-CPI-2M vaccinated gerbils (data not shown).

While there is no direct evidence of effect of vaccination on adult worm viability and growth, Bm-CPI-2M vaccination did affect the fertility of female worms, significantly reducing the numbers of developmental stages in female worms. Similarly, vaccination-induced reduced fecundity of L. sigmodontis worms was observed when mice were vaccinated with Ls-CPI-2M or with L. sigmodontis MF (Babayan et al., 2012; Ziewer et al., 2012). We also previously reported that vaccination of gerbil Brugia pahangi excretory/secretory (ES) products reduced the fecundity of female worms in gerbils (Zipperer et al., 2013). In Caenorhabditis elegans, cystatin-like inhibitor CPI-2a deletion mutants showed that CPI-2a has an essential regulatory role during oogenesis and fertilization (Hashmi et al., 2006). It is possible that Bm-CPI-2 could have a similar role in the embryogenesis of B. malayi. Alteration of Bm-CPI-2 function in female worms by vaccination could explain the results seen in these experiments.

In the current experiments, Bm-CPI-2 mutated to alter its immunomodulatory properties proved to reduce adult worm numbers and reduce fecundity. While we did not test the immunomodulatory activity of mutant Bm-CPI-2, the results suggest that this alteration increased the vaccine efficacy of this molecule. Comparison of the immunomodulatory activities of native and mutant Bm-CPI-2 might further explain its relationship to the enhancement of conferred protective immunity. Our findings suggest that Bm-CPI-2M is a potential anti-filarial vaccine candidate but further investigations are necessary to explore the role of immunomodulation in vaccinology and protective immunity.

Supplementary Material

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Supplementary Fig. S1. Sex ratio of Brugia malayi worms harvested from alum control and Bm-CPI-2M vaccinated gerbils. (A) Experiment 1 (42 days p.i). (B) Experiment 2 (90 days p.i.). The lines represent the mean with S.Ds.

Supplementary Fig. S2. Comparison of total anti-Bm-CPI-2M IgG levels between protected and non-protected gerbils vaccinated with Brugia malayi Bm-CPI-2M from experiment 2. One worm was recovered from the protected gerbil, 24 worms were recovered from non-protected gerbil and 30 worms were recovered from the alum control gerbil. There was no difference in the level of total anti-Bm-CPI-2M IgG between protected and non-protected gerbils vaccinated with Bm-CPI-2M. No anti-Bm-CPI-2M IgG was detected in alum control or pre-immune serum.

Supplementary Fig. S3. Microfilareamia in alum control and Brugia malayi Bm-CPI-2M vaccinated gerbils from experiment 2. There was no significant difference in level of microfilariae between alum control and Bm-CPI-2M vaccinated gerbils. The lines represent the mean with S.Ds.

Supplementary Movie S1. Brugia malayi L3s cultured with peritoneal exudate cells plus gerbil control sera in RPMI media (10× magnification).

Supplementary Movie S2. Brugia malayi L3s cultured with peritoneal exudate cells plus gerbil anti-Bm-CP-2M sera in RPMI media (10× magnification).

Supplementary Movie S3. Brugia malayi L3s cultured with peritoneal exudate cells plus gerbil anti-Bm-CPI-2M sera in RPMI media (40× magnification).

Supplementary Movie S4. Brugia malayi L3s cultured with peritoneal exudate cells plus gerbil anti-Bm-CPI-2M sera in RPMI media (10× magnification).

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Highlights.

  • Brugia malayi cysteine protease inhibitor-2 (Bm-CPI-2) can modulate the host immune response.

  • Vaccination with Bm-CPI-2 does not protect against B. malayi infection in gerbils.

  • Amino acid Asn66 in Bm-CPI-2 is involved in immunomodulation of host immune response.

  • Amino acid Asn66 was mutated to Lys66 in Bm-CPI-2 (Bm-CPI-2M).

  • Bm-CPI-2M vaccination reduces adult B. malayi numbers and its fecundity in gerbils.

Acknowledgments

This work was funded by the National Institutes of Health (NIH), USA -AI07831401. We thank Julie Woody and Jackie Macaluso for their help with this study.

Footnotes

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Associated Data

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Supplementary Materials

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Supplementary Fig. S1. Sex ratio of Brugia malayi worms harvested from alum control and Bm-CPI-2M vaccinated gerbils. (A) Experiment 1 (42 days p.i). (B) Experiment 2 (90 days p.i.). The lines represent the mean with S.Ds.

Supplementary Fig. S2. Comparison of total anti-Bm-CPI-2M IgG levels between protected and non-protected gerbils vaccinated with Brugia malayi Bm-CPI-2M from experiment 2. One worm was recovered from the protected gerbil, 24 worms were recovered from non-protected gerbil and 30 worms were recovered from the alum control gerbil. There was no difference in the level of total anti-Bm-CPI-2M IgG between protected and non-protected gerbils vaccinated with Bm-CPI-2M. No anti-Bm-CPI-2M IgG was detected in alum control or pre-immune serum.

Supplementary Fig. S3. Microfilareamia in alum control and Brugia malayi Bm-CPI-2M vaccinated gerbils from experiment 2. There was no significant difference in level of microfilariae between alum control and Bm-CPI-2M vaccinated gerbils. The lines represent the mean with S.Ds.

Supplementary Movie S1. Brugia malayi L3s cultured with peritoneal exudate cells plus gerbil control sera in RPMI media (10× magnification).

Supplementary Movie S2. Brugia malayi L3s cultured with peritoneal exudate cells plus gerbil anti-Bm-CP-2M sera in RPMI media (10× magnification).

Supplementary Movie S3. Brugia malayi L3s cultured with peritoneal exudate cells plus gerbil anti-Bm-CPI-2M sera in RPMI media (40× magnification).

Supplementary Movie S4. Brugia malayi L3s cultured with peritoneal exudate cells plus gerbil anti-Bm-CPI-2M sera in RPMI media (10× magnification).

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