Can antibody conjugated nanomicelles alter the prospect of antibody targeted therapy against schistosomiasis mansoni?

Abstract

Background: CLA (conjugated linoleic acid)-mediated activation of the schistosome tegument-associated sphingomyelinase and consequent disruption of the outer membrane might allow host antibodies to access the apical membrane antigens. Here, we investigated a novel approach to enhance specific antibody delivery to concealed surface membrane antigens of Schistosoma mansoni utilising antibody-conjugated-CLA nanomicelle technology.

Methodology/principal findings: We invented and characterised an amphiphilic CLA-loaded whey protein co-polymer (CLA-W) as an IV injectable protein nanocarrier. Rabbit anti-Schistosoma mansoni infection (anti-SmI) and anti-Schistosoma mansoni alkaline phosphatase specific IgG antibodies were purified from rabbit sera and conjugated to the surface of CLA-W co-polymer to form antibody-conjugated-CLA-W nanomicelles (Ab-CLA-W). We investigated the schistosomicidal effects of CLA-W and Ab-CLA-W in a mouse model of Schistosoma mansoni against early and late stages of infection. Results showed that conjugation of nanomicelles with antibodies, namely anti-SmI, significantly enhanced the micelles' schistosomicidal and anti-pathology activities at both the schistosomula and adult worm stages of the infection resulting in 64.6%-89.9% reductions in worm number; 72.5-94% and 66.4-85.2% reductions in hepatic eggs and granulomas, respectively. Treatment induced overall improvement in liver histopathology, reducing granuloma size and fibrosis and significantly affecting egg viability. Indirect immunofluorescence confirmed CLA-W-mediated antigen exposure on the worm surface. Electron microscopy revealed extensive ultrastructural damage in worm tegument induced by anti-SmI-CLA-W.

Conclusion/significance: The novel antibody-targeted nano-sized CLA delivery system offers great promise for treatment of Schistosoma mansoni infection and control of its transmission. Our in vivo observations confirm an immune-mediated enhanced effect of the schistosomicidal action of CLA and hints at the prospect of nanotechnology-based immunotherapy, not only for schistosomiasis, but also for other parasitic infections in which chemotherapy has been shown to be immune-dependent. The results propose that the immunodominant reactivity of the anti-SmI serum, Schistosoma mansoni fructose biphosphate aldolase, SmFBPA, merits serious attention as a therapeutic and vaccine candidate.

Fig 1. Schematic illustration of fabrication of CLA-whey protein nanomicelles (CLA-W) and antibody-CLA-W conjugated nanomicelles (Ab-CLA-W).

CLA-W nanomicelles were prepared via carbodiimide (EDC) coupling reaction of the primary amine (-NH₂) of whey protein and the carboxyl group (-COOH) of CLA through amide bond formation. Antibody-succinate (Ab-Su) was formed by addition of succinic anhydride to purified anti-SmI/anti-SmAP IgG antibodies. The formed Ab-Su was activated by EDC.HCl/K-Oxyma to form an Oxyma active ester intermediate that could covalently couple with the primary amines of CLA-W using succinic anhydride as a linker to produce the antibody-succinate whey-CLA copolymer (Ab-CLA-W), the antibody-CLA-W conjugated nanaomicelles.

Fig 2. 10% SDS-PAGE gel of anti-SmI and anti-SmAP IgG antisera and their purified forms.

Coomassie blue-stained 10% SDS-PAGE gel of rabbit anti-SmI and anti-SmAP antisera under non-reducing conditions (lanes 1, 7) and reducing conditions (lanes 2, 8). Purified anti-SmI and anti-SmAP IgG antibodies under non-reducing conditions (lanes 3, 5) and reducing conditions (lanes 4, 6). Lane M, protein molecular weight marker; lanes 1 and 2, rabbit anti-SmI serum; lanes 3 and 4, purified rabbit anti-SmI IgG antibodies; lanes 5 and 6, purified rabbit anti-SmAP IgG antibodies; lanes 7 and 8, rabbit anti-SmAP serum. Anti-SmI and anti-SmAP IgG antibodies were purified from their respective sera in lanes 1, 2, 7, 8 utilising Protein G Sepharose 4 Fast Flow resin. Purified IgG protein bands, non-reduced IgG dimer (at >240 kDa) and reduced IgG heavy and light chains (at 55 and ~28 kDa) are arrowed in red and green, respectively.

Fig 3. FTIR spectra of CLA, whey protein, and CLA-W conjugate (CLA-W nanomicelles).

Line graphs of FITR spectra of composition of CLA (blue), whey (orange), and CLA-W nanomicelles (grey).

Fig 4. TEM of the free CLA-W nanomicelles and Ab-CLA-W nanomicelles.

Transmission electron microscope (TEM) micrographs (x3000) of: (A) free CLA-whey (CLA-W) nanomicelles showing rounded smooth surfaces with medium sized particles ranged from 104.70–130.76 nm; (B) anti-SmI-CLA-W nanomicelles showing rounded smooth surfaces with sizes ranged from 251.86–275.93 nm; (C) anti-SmAP-CLA-W nanomicelles showing rounded smooth surfaces with an sizes ranged from 215.54–288.81 nm.

Fig 5. Antigenic reactivities of purified, CLA-W conjugated IgG antibodies.

(A) Indirect membrane immunofluorescence assay of lung schistosomula recovered from infected, untreated mice and incubated with: anti-SmI antiserum showed IF intensity of 4+ (i), purified, CLA-W conjugated anti-SmI IgG antibodies showed IF intensity 3+ (ii), anti-SmAP antiserum showed diminished staining (few spots) immunofluorescence labelling on their surfaces (iii), purified, CLA-W conjugated anti-SmAP IgG antibodies showed diminished staining (few spots) immunofluorescence labelling on their surfaces (iv), normal rabbit serum showed no IF staining (v) (x400) Scale bar = 50 μm. (B) Non-reducing 10% (Bi) and 8% (Bii) Western immunoblots of adult worm homogenate probed with: anti-SmI antiserum (Bi, lane 1); purified, CLA-W conjugated anti-SmI IgG antibodies (Bi, lane 2) showing reactivity at ~ 33 kDa; anti-SmAP antiserum (Bii, Lane 1); purified anti-SmAP IgG antibodies (Bii, lane 2) showing reactivity at ~120 kDa (blue arrows); normal rabbit serum (Bi and Bii, lane 3).

Fig 6. Immunofluorescent staining of S. mansoni adult worms recovered from mice treated with unconjugated CLA-W nanomicelles.

The surface of S. mansoni adult worms perfused form mice that had received CLA-W nanomicelles and from control untreated mice was probed with anti-SmI antiserum (i), anti-SmAP (ii) or a serum from a naïve rabbit (iii), followed by FITC-goat anti-rabbit IgG antibodies. (i) A, adult male and female worms from untreated mice showed IF intensity of 1+; B, Adult male and female from mice received CLA-W nanomicelles on days 5 & 6 PI showed IF intensity of 1+; C and D, adult male (C) and female (D) worms from mice received CLA-W nanomicelles on days 35 & 36 PI showed IF intensity of 4+. (ii) A, paired male and female worms from untreated mice showed IF intensity of 1+; B, paired adult worms from mice received CLA-W nanomicelles on days 5 & 6 PI showed IF intensity of 1+; C, adult male and female worms from mice received CLA-W nanomicelles on days 35 & 36 PI showed IF intensity of 4+. (iii) A and B, adult male (A) and female (B) worms from mice received CLA-W nanomicelles on days 5 & 6 PI showed no IF staining; C and D, adult male (C) and female (D) worms from mice received CLA-W nanomicelles on days 35 & 36 PI showed no IF staining. Scale bar = 100 μm.

Fig 7. SEM of S. mansoni adult worms recovered from untreated mice and mice treated with anti-SmI-CLA-W nanomicelles.

Scanning electron micrographs (SEM) of S. mansoni adult worms recovered from an infected, untreated mouse (i, A-G) and from a mouse that had been treated with anti-SmI-CLA-W nanomicelles on days 35 & 36 PI against the adult stage, SGIVb (ii, A-J). (i) A, SEM showing the normal surface topography of an adult male worm with round to oval, intact oral sucker and ventral suckers (x75); B, normal anterior end of a male worm showing rounded oral and ventral suckers (OS, VS) with intact surface (x250); C, dorsolateral tegumental surface of the mid-body region showing tubercles (T) of uniform size and distribution covered with apically situated spines (S) and intact surface in between the tubercles with evident ridges (R) (x2,500); D, normal adult female with elongated smooth body and intact tegument (x75); E and F, mid body region of a female worm showing normal smooth ridged tegument with conspicuous minute sensory papillae (SP) (x700 and x2,500, respectively); G, normal posterior end of female worm showing tegument covered by short spines interspersed with sensory papillae (SP) (x2,000). (ii) A, adult treated male showing extensive deformity with body tegumental oedema (x50); B, anterior end of a treated male worm showing distortion of the oral sucker (OS) and retraction (arrow) of the ventral sucker (VS) (x800); C, mid-body region of a treated male showing dorsal tubercles (T) with diminuted spines and deepening (D) of the furrows (x2,200); D, mid-body region of a treated male showing sloughed tubercles (T) with exposure of subtegumental tissues and attached leuckocytes (arrows) (x2,200); E, tegumental surface between the tubercles (T) of a treated male’s mid-body region showing complete loss of spines (x1,500) with deepening of the furrows (D); F, a treated S.mansoni worm couple’ mid body region with the female showing extensive swelling of the tegument and longitudinal corrugations (x450); G, the mid-body region of a treated female showing swelling and peeling of the tegument (x550); H and I, the mid-body region of a treated female showing oedema and sloughing of the tegument with exposure of the subtegumental tissues giving honey comb appearance (x300 and x6,500,respectively); J, posterior end of a treated female showing oedema and complete loss of spines (x2,000). Scale bar = 200μm (iA, iD, iiA), 100μm (iB), 50μm (iiF), 20μm (iE, iiB, iiG), 10μm (iB, iF, iG, iiC, iiD, iiE, iiH, iiJ), 5μm (iiI).

Fig 8. TEM of S. mansoni adult worms recovered from infected, untreated mice.

Transmission electron micrographs (TEM) show normal tegument, gastrodermis and mature vitelline cells of S.mansoni adult worms recovered from an infected, untreated mouse. A, normal tegumental ultrastructure of adult male showing Syncytia (Syn), basal membrane (Bm), invaginations of basal membrane (Inv), circular muscle (Cm), longitudinal muscle (Lm), diagonal muscle (Dm), discoid bodies (arrow head), multilamellar bodies (dotted arrow) and microtubule-lined cytoplasmic channels (arrow) (x4,000); B, normal subtegumental cyton of a male showing euchromatic nucleus (euN), nucleolus (No), discoid bodies (arrow head), multilamellar bodies (dotted arrow), free ribosomes (R) and mitochondria (mt) (x4,000); C, normal gastrodermis of adult male showing syncytia (Syn), nucleus (N), long cytoplasmic extensions (arrow) and muscle layer (M) (x 2,500); D, normal mature vitelline cell of adult female showing the characteristic vitelline droplets (Vd) and lipid droplets (Ld) (x3,000). Scale bar = 2.0μm.

Fig 9. TEM of S.mansoni adult worms recovered from mice treated with anti-SmI-CLA-W nanomicelles.

Transmission electron micrographs (TEM) show the effect of anti-SmI-CLA-W conjugated nanomicelles treatment on the tegument, gastrodermis and mature vitelline cell of S. mansoni adult worms recovered from a mouse that had been treated on days 35 & 36 PI against the adult stage, subgroup IVb. A, tegumental ultrastructure of adult male S. mansoni showing erosion of the tegument (Er), extensive vacuolization (V) in syncytia and swollen mitochondria (mt) (x2,000); B, tegumental ultrastructure of adult male S. mansoni showing extensive vacuolization (V) in syncytia and disruption of the subtegumental ultrastructure architecture (x2,500); C, tegumental ultrastructure of adult female showing extensive muscle lysis (L), whorled myelin figures (circle) and swollen mitochondria (mt) (x4,000); D, Subtegumental cyton of adult male showing vacuolization (V) of cytoplasm, irregularity in the nuclear membrane and swollen mitochondria (mt) (x2,500); E, The gastrodermis of adult male showing oedema and vacuolization (V) in syncytial layer with decreased cytoplasmic extensions (black arrow) (x2,500); F and G, Mature vitelline cell of adult female showing vacuolization (V) and fusion of the vitelline droplets (Vd) (x3,000 and x5,000, respectively). Scale bar = 1 μm in (D) and (G), and 2.0 μm in all other micrographs.

Fig 10. S. mansoni egg oogram pattern and hepatic granulomas sizes in treated mice.

(i) Graph showing oogram pattern of S. mansoni eggs in the small intestines of mice received different treatment schedules on days 35 & 36 PI. Columns represent mean relative percentages of eggs in their progressive stages of live egg maturity (immature, mature) and dead eggs. (ii) Graph showing mean sizes of S. mansoni egg granulomas (μm) in livers of mice for each subgroup received different treatment schedules on days 5 & 6 PI (against the schistosomula stage) and 35 & 36 PI (against the adult worm stage). Error bars represent standard deviations of 6 mice per subgroup. *** P<0.001.

Fig 11. Effect of CLA-W nanomicelles and antibody-CLA-W-nanomicelles treatments on the liver histopathology of S.mansoni infected mice.

H&E stain and Masson’s trichrome stained Liver sections from untreated mice, SGIb (A-D), mice treated on days 35 & 36 PI with: CLA-W nanomicelles, SGIIb (E-H), anti-SmI-CLA-W nanomicelles, SGIVb (I-L) and anti-SmAP-CLA-W nanomicelles, SGIVb (M-P), showing: (A, B) Multiple granulomas of different shapes and sizes with disturbance in hepatic architecture. (C) Hepatocytes showing vacuolar degeneration (vd) with focal necrosis (n) and marked Kupffer cells hyperplasia with deposition of bilharzial pigments (arrows). (D) Multiple granulomas with evident peri-granulomatous hepatic fibrosis. (E, F) Multiple granulomas of different shapes and sizes with disturbance in hepatic architecture. (G) Hepatocytes showed bilharzial pigmentation, vacuolar degeneration (vd) and sinusoidal dilatations (sd). (H) Hepatic granuloma with moderate hepatic fibrosis. (I, J) Scanty small hepatic granulomas with central hyalinized eggs. (K) Mild degree of hepatocytes swelling with residual bilharzial pigments in hyperplastic kupffer cells. (L) Very small hepatic granuloma with minimal hepatic fibrosis. (M, N) Few, small hepatic granulomas surrounding dead calcified eggs with mild inflammatory reaction. (O) Mild degree of hepatocytes swelling with residual bilharzial pigments in hyperplastic kupffer cells. (P) Small hepatic granuloma with mild hepatic fibrosis. Scale bar = 500μm (i), 200μm (ii and iv), 50μm (iii). H&E stain (i, ii, iii); Masson’s trichrome stain (iv).

Fig 12. Liver histopathology of mice treated against lung schistosomula (SGa) and antibodies-alone against adult worms (SGIIIb&SGVb).

(i) A and B, multiple granulomas of variable shapes and sizes with abnormalities in hepatic architecture (SGIIIb and SGIIa); C, small hepatic granulomas surrounding dead calcified eggs (SGIVa). (ii) D, multiple granulomas of variable shapes and sizes with disturbance in hepatic architecture (SGIIIb); E, cellular granulomas with viable S. mansoni eggs recovered from (SGIIIa); F, a small hepatic granuloma surrounding dead calcified egg (SGIVa). (iii) G and H, hepatocytes showing vacuolar degeneration (vd) with focal necrosis (n) and sinusoidal dilatation (sd) (SGVb and SGVIa); I, mild degree of hepatocytes swelling, Kupffer cell hyperplasia (SGIVa). (iv) J, multiple granulomas variable in size and shape with evident hepatic fibrosis (SGIIIb); K, reduced peri-granulomatous fibrosis of liver sections (SGVIa); L, hepatic granuloma with mild hepatic fibrosis (SGIVa). Scale bar = 500μm (i), 200μm (ii and iv), 50μm (iii).