The Study On Long-acting Drug For The Prevention Of Schistosoma Japonicum Infection

Schistosomiasis is a zoonotic parasitic disease, known to represent a seriousthreat to human health. Currently, there are600million people rfom74countries andregions throughout the world at risk of schistosome infection, with nearly200millionpeople already infected. There are six types of schistosomes capable of humanparasitization, although only Schistosoma japonicum is prevalent in China.Schistosomiasis has the characteristic of "concomitant immunity". In such cases,schistosome-speciifc immunity is immediately lost when the parasite is removed rfomthe host, which is subsequently susceptible to re-infection by contact with infectedwater. There are a number of schistosome reservoir hosts and many natural epidemicfoci in the natural world. Food production practices and lifestyle mean that thepopulation in epidemic areas is repeatedly exposed to infected water, resulting in ahigh incidence of schistosome re-infection. Granuloma, ifbrosis, and liver dysfunction,as well as chronic illness and death are the major outcomes of schistosomiasis, as aresult of continuous inflammation caused by eggs deposited in liver tissue. Damage tohuman health could be effectively reduced by eradication of the schistosomulumimmediately atfer entry into the host, or by preventing development into adult wormswhich lay eggs.At present, the development of vaccines to prevent schistosome infection areseriously hindered by the complicated mechanism of immune evasion by theschistosome. Thus, the control of schistosomiasis prevalence still depends mainly onchemotherapeutic approaches. Praziquantel is the preferred drug for schistosomiasistreatment; however, due to its lack of eiffcacy in eradicating the schistosomulum, thisdrug cannot be used prophylactically. In the early1980s，the effect of artemisinin andits derivatives against schistosome infection was discovered by researchers in China.In contrast to praziquentel, artemisinin effectively eradicated the schistosomulum, andcould be used for the prevention and early treatment of schistosome infection. It isreported that, at equivalent doses，the effect of artesunate (AS) on the schistosomulum is higher than that of artemether and other derivatives. The short half-life of AS makesrepeat dosing in the short-term a requirement for the prevention and treatment ofschistosome infection, which increases the treatment cost and reduces patientcompliance. If the half-life of AS could be extended, a prolonged effectiveconcentration could be achieved in vivo, which would improve the therapeuticeiffcacy of AS and could also prevent schistosome infection. In the absence of aneffective prophylactic vaccine, regular administration of long-acting AS to thepopulation and livestock within endemic areas would provide an ideal strategy for thecontrol of schistosome infection.AS，and derivatives of this small compound, are suitable for clearance byglomerular ifltration. Theoretically, increasing the hydro dynamic volume of AS (bymethods such as coupling AS with some kind of macro molecular protein) wouldprolong its half-life and the effective drug concentration of AS in vivo could beprolonged to achieve the prevention of schistosome infection. The aim of this studywas to extend the half-life of AS in vivo by chemically coupling it with the lowimmunogenieity protein molecule XTEN’, to obtain a long-acting drug which couldbe used for preventing schistosome infection of different reservoir hosts and to controlthe prevalence of schistosomiasis.Part I The expression and puriifcation of the GST-XTEN1fusion proteinA gene of1,368bp, encoding the XTEN’ protein (41.858kDa) was designedaccording to Volker Schellenberger’s strategy and synthesized by ShanghaiG?eneray Biotechnology Co.(China). A recombinant expression plasmidXTEN’-pET41a was constructed successfully by subcloning the DNA rfagmentencoding the XTEN’ protein into the BamHl and Sail restriction enzyme sites of theexpression plasmid pET41a. Transformants containing the recombinant expressionplasmid XTEN’-pET41a were inoculated into LB medium and protein expressioninduced by IPTG. A soluble GST-XTEN’ tfision protein dimer (approximately120kDa) was expressed successifilly under conditions optimized for induction oftransformants with IPTG at the stationary growth phase for4h at37°C. The solublerecombinant protein GST-XTEN’ tfision protein dimer was puriifed rfom expression products by Glutathione aiffnity chromatography. The purity of the recombinantprotein GST-XTEN’ exceeded95%.Part II The preparation and characterization of the AS-GST-XTEN1conjugateThe AS-GST-XTEN’ conjugate was prepared by coupling the carboxyl group ofthe long arm of artesunate with the rfee amino group of the GST-XTEN’ proteinthrough a mixed anhydride method. The characteristic absorption peaks of AS，GST-XTEN’ and the AS-GST-XTEN’ conjugate were determined by Ml wavelengthspectrophotometric scanning. The AS-GST-XTEN’ conjugate exhibited acharacteristic ultraviolet absorption peak at270nm, which is different to that of AS(maximum absorption wavelength,260nm) and GST-XTEN’ protein (maximumabsorption wavelength,280nm), thus indicating successful preparation of theAS-GST-XTEN’ conjugate. The coupling rate was1:506; that is，an average of onemolecule of GST-XTEN’ protein was coupled with506AS molecules.The schistosomula were cultured in24-well plates at37°C with5%CO2. AS orthe AS-GST-XTEN’ conjugate were added at different concentrations into the culturemedium, at the same time. Controls comprised the addition of RPMI1640or5%NaHC〇3. Schistosomula death was observed atfer20min，4h，24h，and48h underan inverted microscope. The AS-GST-XTEN’ conjugate exhibited insecticidal effects,although its dosage was greater than that of artesunate. The insecticidal effect of theconjugate was also slower than that of AS. The different concentration gradients ofAS and the AS-GST-XTEN’ conjugate were used to calculate the LD50values of thetwo drugs. To achieve equivalent insecticidal effects during the same time, the dose ofthe AS-GST-XTEN’ conjugate was approximately4-times greater than that ofartesunate.ICR mice were injected subcutaneously with the AS-GST-XTEN’ conjugate (3jig specific antigen per mouse, without any immunological adjuvants) once per weekfor six weeks. Serum was isolated rfom blood samples collected rfom the caudal veinprior to immunization, one week atfer the forth inoculation (day29) and one weekatfer the sixth inoculation (day42). The serum anti-AS-GST-XTEN’ conjugate IgGtiter of the immunized mice was measured by indirect enzyme-linked immunosorbent assay (ELISA) with the recombinant tfision protein used as the capture antigen. Theserum anti-AS-GST-XTEN’ conjugate IgG titer was1:1600-1:3200at four weeks postimmunization and1:3200-1:6400at six weeks post immunization, which indicatedweak immunogenieity of the recombinant GST-XTEN’ tfision protein.Sixty-six mice were randomly divided into the AS group and theAS-GST-XTEN’ conjugate group, and were injected with AS (26.8mg/kg) and theAS-GST-XTEN’ conjugate (12.05mg/kg) intravenously (tail vein). Serum wasisolated rfom blood samples collected at2min，15min，30min，1h，2h，5h，12h,24h,51h,72h and97h atfer drug administration. The serum concentration of AS wasdetermined by a sandwich ELISA and was shown to reach a peak value rapidly at2min atfer injection, with a half-life of approximately30min. These data are consistentwith previous reports. However, although the serum concentration of theAS-GST-XTEN’ conjugate reached a peak more slowly at15min post-injection, thehalf-life was signiifcantly extended to2h atfer injection. Thus, the AS-GST-XTEN’conjugate was shown to have a longer half-life than that of AS.Part III The effect of the AS-GST-XTEN，conjugate on the treatment andprevention of schistosome infectionTo observe the effect of the AS-GST-XTEN’ conjugate against theschistosomulum in vivo,30ICR mice were divided equally into three groups (AS，AS-GST-XTEN’ and Control). Each mouse was infected with35S. japonicumcercaria by abdominal skin penetration. At day7atfer schistosome infection (thelung-stage), each mouse was injected with AS (150mg/kg) or the AS-GST-XTEN’conjugate (600mg/kg based on AS content) intravenously (tail vein). A group ofuntreated mice represented the Control. Mice were sacriifced at day35post-infection,and enumeration of the S. japonicum adult worms in the veins showed that the effectof the AS-GST-XTEN’ conjugate against the lung-stage somulum of S. japonicum wassimilar to that of AS，with a the worm reduction rate of60%mediated by both drugs(尸<0.05).To investigate the prevention of schistosome infection,70ICR mice were dividedrandomly into seven groups (10mice per group), and drugs were administered intravenously (tail vein). Groups1，2and3were injected with AS (150mg/kg) andGroups4，5and6were injected with the AS-GST-XTEN’ conjugate (600mg/kg basedon AS content). Group7(control) were untreated. Mice were infected with S.japonicum cercaria in a simulated natural infection environment as follows: Groups1，4and7at day7post-injection; Groups2and5at day14post-injection; Groups3and6at day21post-injection. All mice were sacriifced by cervical dislocation at day35post-infection and worms in veins were counted to calculate the worm reduction rate.The results showed that the average worm burden was49.00土0.56in the Controlgroup (Group7)，while the average worm burdens of Groups1，2and3(infected withcercariae at days7，14and21，respectively, post-injection with AS) were48.60士0.50，48.30士0.73，48.60士0.65，respectively. There was no significant difference in the wormburden (all P>0.05) detected in Groups1，2and3compared with Group7. Theaverage worm burdens of Groups4，5and6(infected with cercariae at days7，14，and21，respectively, post-injection with the AS-GST-XTEN’ conjugate) were32.90士0.53，48.80士0.66，and48.90士0.69，respectively. The worm burden of Group4wassigniifcantly lower than that of Group7(P <0.05)，with a worm reduction rate of32.8%. However, there were no significant differences between the worm burdens ofGroups5and6compared with Group7(all P>0.05).ConclusionIn the present study, a recombinant plasmid XTEN’-pET41a was constructed.The recombinant GST-XTEN’ ifision protein was successfully expressed and purified,and a conjugate (AS-GST-XTEN1) of artesunate and the GST-XTEN’ protein wasprepared by chemical methods. The AS-GST-XTEN’ conjugate exhibited lowerimmunogenieity and exerted effective insecticidal activity against schistosomulumboth in vivo and vitro. Furthermore, the half-life of the AS-GST-XTEN’ conjugatewas prolonged in vivo and preliminary investigations indicated the eiffcacy in theprevention of schistosome infection.