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WO1997033610A1 - ADN CODANT POUR LA GLUTATHION-S-TRANSFERASE DE 28 kDa DU SCHISTOSOMA MANSONI ET SES UTILISATIONS - Google Patents

ADN CODANT POUR LA GLUTATHION-S-TRANSFERASE DE 28 kDa DU SCHISTOSOMA MANSONI ET SES UTILISATIONS Download PDF

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WO1997033610A1
WO1997033610A1 PCT/US1997/003977 US9703977W WO9733610A1 WO 1997033610 A1 WO1997033610 A1 WO 1997033610A1 US 9703977 W US9703977 W US 9703977W WO 9733610 A1 WO9733610 A1 WO 9733610A1
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plasmid
dna
mice
cells
protein
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WO1997033610A9 (fr
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Stephen G. Kayes
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University of South Alabama
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University of South Alabama
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C12N9/1088—Glutathione transferase (2.5.1.18)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates generally to the fields of parasitology and molecular biology. More specifically, the present invention relates to a naked DNA vaccine for parasitic diseases.
  • Schistosomiasis is a disease that affects over 250 million people worldwide, primarily in underdeveloped countries and approximately one percent of those who contract schistosomiasis will die annually from hemorrhage and/or liver failure.
  • the causative agent is the human blood fluke, Schistosoma manson i which lives in the inferior mesenteric venules. In these venules, the male and female worms live, mate, and produce large numbers of eggs each day which ultimately accumulate in the liver and elicit a characteristic granulomatous inflammation.
  • the ensuing disease is characterized by hepatosplenomegaly, portal hypertension and esophageal varices, the latter often being fatal.
  • praziquantel ® and oxamniquine® are used to treat schistosomiasis but reinfection occurs with return to fresh water (where the snail intermediate hosts of S. mansoni live) and there is evidence accumulating that the worms are becoming resistant to these agents .
  • lymphocyte proliferation assay i.e., the lymphocyte transformation assay or LTA
  • PHA and Con A T cell mitogens
  • Schistosoma mansoni egg antigens 21 -24.
  • DNA when taken up by skeletal muscle, remains circular and neither integrates into chromosomal DNA nor replicates (5, 6).
  • Naked DNA techniques 1 1
  • intramuscular injection of plasmid DNA in particular have been used to immunize (i.e., vaccinate) experimental animals, and direct injection has been shown to elicit both cellular and humoral immune responses ( 1, 8- 10).
  • Mice have been successfully immunized by intramuscular injection of plasmid DNA carrying an influenza gene as determined by their ability to survive a lethal challenge infection (9, 10).
  • Using the same polynucleotide vaccine in mice revealed that 1 ) a single injection elicited both cellular and humoral responses that persisted for 1 year and 2) the cellular response was associated with the CD8+ subpopulation of T cells (i.e., killer T cells) (12).
  • mice were given plasmid DNA injections by one of three routes (iv, ip, or im) and subjected to an influenza virus- challenge.
  • gene vaccination resulted in 60% protection which was attributed to use of a gene from a heterologous virus (i.e., a virus related to the influenza virus that normally infects chickens but differing by 16% in amino acid sequence).
  • Mice which have been vaccinated with plasmid DNA encoding a bovine herpes glycoprotein have gone on to make significant amounts of antiglycoprotein antibody and the sera from these mice contained significant amounts of viral neutralizing activity as compared to mice receiving the plasmid platform lacking the herpes gene (28).
  • the prior art is deficient in the lack of effective means of treating Schistosomiasis using naked DNA vaccination technology.
  • the present invention fulfills this longstanding need and desire in the art.
  • the present invention discloses several eukaryotic expression plasmids containing genes derived from S. mansoni that serve as the basis of a polynucleotide vaccine.
  • no adjuvants are required, no infectious agents (either killed or attenuated) are introduced into the patient during the immunization protocol, and no exogenous contaminants of any kind are introduced which could elicit spurious immune responses.
  • gene products produced by skeletal muscle are presented to the immune system in such a way as to elicit cell-mediated immunity which makes this novel technique ideally suited for development into a human vaccine.
  • the present invention uses naked DNA vaccine technology to establish the feasibility of preventing or ameliorating the effects of infection with the human intravascular blood fluke, Schistosoma mansoni.
  • the present invention characterizes both cellular and humoral immune responses of mice vaccinated with
  • Experimental development of naked DNA vaccination technology is important, not only for the control of the parasite causing schistosomiasis in 250 million people, but for establishing a new way to vaccinate against almost any infectious helminth in manner that does not require live or attenuated organisms or the addition to the vaccine of noxious chemicals in the form of adjuvants such as peanut oil or alum.
  • the present invention determines ( 1 ) the effect of time and dose on the development of the cellular immune response of mice vaccinated with naked DNA encoding Schistosoma mansoni glutathione S-transferase; (2) the effect of time and dose on the development of the humoral immune response of mice vaccinated with naked DNA encoding Schistosoma mansoni glutathione S-transferase; (3) the effect of the H-2 haplotype on the cellular and humoral immune responses of mice vaccinated with naked DNA encoding Schistosoma mansoni glutathione S-transferase.
  • the present invention uses selected strains of inbred mice which are known permissive hosts for the entire life cycle of S. mansoni.
  • lymphocyte transformation assays in response to parasite glutathione S-transferase and granuloma formation around glutathione S-transferase-coated beads are monitored while humoral immune responses (i.e. , antibody production) are evaluated using a glutathione S-transferase specific enzyme-linked immunosorbent assay (ELISA).
  • ELISA glutathione S-transferase specific enzyme-linked immunosorbent assay
  • composition of matter comprising a naked DNA vaccine for the parasitic worm, Schistosoma mansoni or human blood fluke.
  • novel plasmids and expression vectors for use in the methods of the present invention.
  • a method of protecting against Sch is tos o m a manson i comprising administering to an animal in need of such treatment a pharmacologically effective dose of a naked DNA vaccine.
  • Figure 1 shows a schematic drawing of the deletion of CMV promoter and replacement with ⁇ S A pro m oter .
  • Inset miniprep of pRc ⁇ SA cut with (S)ma I and (P)vu II.
  • Figure 2 shows the histochemical localization of E. coli ⁇ gal activity in mouse skeletal muscle 3 weeks after injection of 100 ⁇ l saline of b) saline with 50 ⁇ g pRSV-LacZ DNA. Orig. 400x.
  • FIG. 4 shows that SmGST expression by HEK 293 cells 48 hours after CaPO4 transfection.
  • (-) no DNA.
  • CDNB; chloro-dinitrobenzene used as substrate; n number of dishes assayed.
  • Figure 5 shows the splenic mass 8 weeks after no injection, intramuscular injection of 10 ⁇ g pSmGST3, 50 ⁇ g pSmGST3, or a subcutaneous injection of 25 ⁇ g of pure SmGST mixed with alum .
  • pSmGST3 and pCMV-SmGST3 are used interchangeably and are of equal rank in identification.
  • 130%- line significance.
  • n number of spleens.
  • Figure 6 shows an electrophoretogram showing purification of a 28 kDa protein encoding the S . manson i glutathione S-transferase.
  • Figure 6A shows bacteria transduced with pCMV-GST3.
  • Figure 6B shows control bacteria transduced with pCMV-Lux encoding an irrelevant protein.
  • Lane 1 molecular weight markers X 1000;
  • Lane 2 bacerial lysate (starting material) which was applied to a glutathione agarose affinity column;
  • Lane 3 last wash prior to eluting the column;
  • Lane 4 shows the concentrate of eluate from glutathione-agarose affinity column.
  • Figure 7A and 7B show the nucleotide sequence of plasmids pRc/ASK8-SmGST3 and pCMV-SmGST3.
  • Figure 8 shows the SDS-PAGE ( Figure 8A) and Western Blot (Figure 8B) of uninfected or Bac-SmGST-3-infected Sf21 cells.
  • Figure 8B The gel in Figure 8A was electroblotted to a nitrocellulose membrane and immunostained with polyclonal rabbit anti-S.
  • Figure 9 shows a SDS-PAGE of various stages during the isolation procedure of recombinant SmGST-3 protein from Bac-
  • Figure 10 shows a Western Blot confirming the immunoreactive property of the 28 kDa protein band following concentration from pooled fractions 4 through 12 depicted in
  • the present invention provides a vaccine comprising a non-infectiou s , non-inte grating DNA sequence encoding Schistosoma mansoni glutathione S-transferase.
  • the DNA comprises a plasmid encoding a protein, polypeptide or peptide which is operably linked to a promoter.
  • the plasmid is pCMVSmGST-3.
  • the plasmid is selected from the group consisting of pBsCMV- 16.4 DNA and pRc/ASK8-SmGST3.
  • the present invention is also directed to a method of protecting against infection by Schistosoma mansoni comprising the step of: administering to an animal in need of such treatment a pharmacologically effective dose of a vaccine composition comprising a non-infectious, non-integrating DNA sequence encoding Sch isto soma manson i glutathione S -transferase .
  • the vaccine composition comprises a plasmid encoding a protein, polypeptide or peptide which is operably linked to a promoter.
  • Representative examples of plasmids useful in this method of the present invention include pCMVSmGST-3, pBsCMV- 16.4 DNA and pRc/ASK8-SmGST3.
  • the plasmid is administered in an animal in a dose of from about 25 ⁇ g to about 200 ⁇ g.
  • the plasmid is administered into muscle although it is possible it could be adminstered into another site in the body.
  • the plasmid would be administered between 1 and 6 times.
  • the present invention is also directed to a method of developing cell mediated immunity to glutathione-S-transferase of Schistosoma mansoni comprising the step of: administering to an animal in need of such treatment a immunologically effective dose of a plasmid of the present invention.
  • Representative examples of plasmids useful in this method of the present invention include pCMVSmGST-3 , pBsCMV- 16.4 DNA and pRc/ASK8-SmGST3.
  • plasmid is administered in an animal in a dose of from about 25 ⁇ g to about 200 ⁇ g.
  • the plasmid is administered into muscle although it is possible it could be adminstered into another site in the body.
  • the plasmid would be administered between 1 and 6 times.
  • a technique for injecting naked DNA directly into skeletal muscle using a reporter plasmid in which the lacZ gene was inserted 3' to an RSV promoter in a eukaryotic expression plasmids engineered was used.
  • muscles were removed, frozen sections prepared, the sections stained for ⁇ -gal activity and the histochemical localization was compared to control muscles injected with saline.
  • All lacZ DNA-injected muscles stained intensely indicating the presence of the plasmid and expression of the bacterial gene by the mouse muscle cells while all controls were completely devoid of reaction product (See Figure 1 ).
  • the cDNA encoding the SmGST-3 isoenzyme was obtained.
  • the cDNA was excised from a pGEX plasmid and ligated into a eukaryotic expression plasmids 3' to an immediate early CMV promoter.
  • HEK 293 cells in tissue culture were transfected by the CaPO4 method. Plates at the time of transfection were approximately 50% of confluency and after transfection were allowed to incubate at 37°C/5% CO2 in air for another 48 hr. At this time the cells were confluent which indicates that the cells tolerated the foreign DNA well.
  • Soluble extracts of the monolayers were shown (Figure 2) to overexpress GST activity when using chlorodinitrobenzene (CDNB) as the electrophilic substrate in the assay described by Habig and Jakoby (20) as compared to cells transfected with an irrelevant plasmid (which was also determined to be expressed, but is not shown in Fig. 2).
  • CDNB chlorodinitrobenzene
  • BALB/c mice have been vaccinated with various quantities of either pCMVSmGST-3 or an irrelevant plasmid (pRSV- Lux) and after two weeks the mice were infected with 200 cercariae of S. mansoni.
  • FIG. 3 based on splenic mass increase (which correlates with lymphocyte proliferation in the white pulp) illustrates that mice receiving the pCMVSmGST-3 DNA are affected by the treatment.
  • Glutathione S-transferases (EC 2.5.1.18) catalyze the conjugation of both endogenous and exogenous electrophiles with the major cellular nucleophile, reduced glutathione (GSH) (29).
  • GSTs are found widely distributed in nature in both invertebrates such as Schistosoma mansoni and vertebrates such as mice, rats and humans ( 18) . Most organisms contain multiple GST isoenzymes that are characterized by distinct but often overlapping substrate specificities ( 18).
  • Adult S. Glutathione S-transferases
  • mansoni contain at least five forms of GSH S-transferase, three of which (designated SmGST- 1 , SmGST-2 and SmGST-3) account for 2-4% of the soluble ⁇ protein in the male worm (19, 20).
  • GSH S-transferases from vertebrates that have been characterized are dimeric proteins composed of either identical (homodimer) or nonidentical (heterodimer) subunits ( 16, 17).
  • the polypeptide subunits are catalytically independent and the quaternary structure is thought to be necessary for enzyme stability ( 17).
  • three cytosolic forms of S. mansoni G S T were purified and found to have very similar catalytic, physicochemical, and immunological properties. However, the most striking feature is that they are catalytically active monomers rather than dimers ( 19).
  • the parasite GST is a 28 kDa protein in contrast to vertebrate GSTs which range from 44 to 50 kDa and b) that the parasite proteins do not immunologically cross react with vertebrate (i.e. host) GSTs which makes the present invention herein feasible because immune responses against parasite GSTs will not immunoneutralize the hosts GSTs.
  • This protein was found to have a Mr of 28 KDa and cDNA encoding the 28 K protein was subsequently cloned in E. coli and the recombinant protein used to immunize rats and BALB/c mice. Good immunoprotection against 5. mansoni in several host species (i.e., approximately 50% in the mice) has been demonstrated using Sm28 and either Freund's Complete or an alum adjuvant (31).
  • This plasmid known as pRc/CMV was obtained from the Invitrogen Corporation and contains the immediate early enhancer-promotor sequence from cytomegalovirus (CMV) , polyadenylation signal and transcription termination sequences from the bovine growth hormone (BGH) gene and SV40 origin.
  • CMV cytomegalovirus
  • BGH bovine growth hormone
  • This plasmid and its host species of permissive E. coli, TOP10F', were used for all subsequent experiments.
  • the CMV promoter was first removed from the pRc/CMV plasmid and replaced with the chicken ⁇ -skeletal actin promoter sequence. This is shown schematically in Figure 1 and was performed as follows.
  • the CMV promoter sequence is flanked 5' by an Nru I site and 3' by a Hind III site just inside the polycloning sequence.
  • a Hind III restriction enzyme (RE) cut was performed on purified pRc/CMV DNA followed by a Klenow-mediated blunt end fill-in of the cut site.
  • the linearized, cut DNA was ethanol precipitated and then cut with Nru I. Because Nru I is a blunt cutter, the pRc backbone was now ready for blunt end ligation.
  • a 6.2 Kb p ⁇ SK fragment of genomic DNA was obtained carrying the chicken a -skeletal actin ( ⁇ SA) gene and its entire promoter (nb: skeletal actin promoters are highly conserved and have been shown to function in mammalian cells).
  • the 675 bp promoter sequence as shown in Figure 1 was contained between an Eco RV and Nae I site.
  • a sequential RE digest was performed and the 675 bp ⁇ SA fragment isolated from a 1 % agarose gel.
  • the 5 Kb pRc plasmid and the 675 bp ⁇ SA fragment were ligated using T4 ligase. The ligation products were then transfected into TOP10F' and 22 colonies were screened for plasmid DNA.
  • plasmids were cut with Sma I which cuts asymmetrically one time within the ⁇ SA sequence and once within the remaining pRc sequence. If the ⁇ SA sequence is in the correct 5'— >3' orientation with respect to the polycloning sites then two bands of 1800 bp and 4000 bp should be seen upon agarose gel electrophoresis. One such clone designated pRc ⁇ SA ⁇ was found (shown in the insert of Figure 1 ) and this orientation was confirmed by cutting with a different RE, Pvu II, which gives fragments of 81 1 , 1065 , 1 196, and 2400 bp.
  • the plasmid DNA was isolated from a liter of bacteria. Glycerol stocks of TOPI OF' containing pRc ⁇ SA8 (pRcASK8 is of equal rank and used interchangeably) were prepared and stored at -80°C. A control plasmid for expression under the control of the ⁇ SA promoter sequence with the promoter sequence inserted in the 3'-->5' orientation was also stocked (pRc ⁇ SA 14) . The next stage was to insert the gene of interest into the polycloning site.
  • the SmGST3 fragment was removed from the pCR3-SmGST3 TA cloning vector using Xba I and ligated into pRcASK ⁇ , which had also been cut with Xba I, using T4 ligase. The ligation products were then transformed into TOP10F' cells.
  • the new construct, designated pRcASK8-SmGST3 was stocked in a glycerol solution and stored at -80°C.
  • a eukaryotic reporter plasmid in which the E. coli ⁇ -galactosidase ( ⁇ - gal) gene is inserted into the polycloning site can be used to verify both properties.
  • ⁇ - gal E. coli ⁇ -galactosidase
  • mice were sacrificed after 1 , 2, or 3 weeks and the injected muscles removed, snap frozen in methyl butanol on dry ice, and 10 ⁇ m frozen sections prepared. The sections were stained with X-gal in the presence of potassium ferriferrocyanide. All muscles injected with the pBsRSV- ⁇ Gal plasmid had scattered myofibers which stained intensely blue- black indicating that the ⁇ -Gal gene product, ⁇ -galactosidase, was being expressed. None of the saline-injected muscles exhibited positive staining ( Figure 2).
  • the cDNA was liberated with Eco RI and using a QIAEX gel extraction kit, the 782 bp fragment was isolated and ligated into an Eco RI site within the polycloning sequence of pB sCMV , a gene transfer vector engineered for eukaryotic gene expression.
  • Orientation was determined by an asymmetrical cut with Mun I which gave two fragments of predicted sizes ( 818 and 4300 bp) while inverse orientation yielded expected fragments of 1538 and 3580 bp. As seen in Figure 3, five colonies contained inserts that were in the correct orientation and three colonies contained inversions. This plasmid was electroporated into electrocompetent DHlOb cells. Based on miniprep analysis of resultant overnight colonies, a liter of bacteria were grown overnight, and expression grade plasmid DNA was isolated by double alkaline lysis followed by double CsCl gradient ultracentrifugation.
  • the resulting DNA was dialyzed against 4 liters of I X TAE buffer, ethanol precipitated and dissolved in 1.0 ml sterile, deionized, distilled H2O. Final DNA and RNA concentrations were determined spectrophotometrically at OD260/280. Two clones, designated 3.7 and 3.9, were prepared in parallel and the final DNA yields were 3.0 and 3.1 mg DNA per ml, respectively .
  • TOP10F' cells carrying the pCMVGST3 plasmid were grown overnight and then collected by centrifugation. The cells were lysed and the soluble fraction collected and applied to a GSH- agarose affinity column to purify the overexpressed protein, SmGST3.
  • a parallel culture of pCMV-Lux expressing the irrelevant protein, luciferase was used as a control. Affinity purified SmGST3 was eluted from the column with 10 mM glutathione. The recovery was monitored by coomassie blue staining of 12% polyacrylamide gel electrophoresis and examination of the 28 kDa band ( Figure 6).
  • HEK 293 cells in tissue culture were transfected by the CaP ⁇ 4 method. Plates at the time of transfection were approximately 50% of confluency and after transfection were allowed to incubate at 37°C in 5% C02 in air for another 48 hours. At this time the cells were confluent which indicated that the cells tolerated the foreign DNA well.
  • CDNB chloro-dinitrobenzene
  • PCR primers were designed to add an Xba I restriction site to both the 5' and 3' ends of the fragment and to add a Kozak consensus sequence that would enhance the binding of the polymerase to the promoter sequence) to the 5' end.
  • the PCR fragment was cloned directly from the thermal cycler into Invitrogen's TA cloning vector, pCR3. This vector is actually designed to serve as a eukaryotic expression vector and the cloned fragment is under the control of the immediate early CMV promoter.
  • This construct was electroporated into DH lOb cells and yielded an 85% cloning efficiency. Tubes of stock bacteria carrying this construct (designated pCR3-SmGST3) were stored at -80°C.
  • mice were injected intramuscularly (im) (vaccinated) with 10 or 50 ⁇ g of either pCMVGST-3 or an irrelevant plasmid (pRSV-Lux, containing the luciferase reporter gene). After two weeks the mice were challenge-infected with 200 cercariae of S. mansoni. An additional group was vaccinated in the conventional way with 25 ⁇ g of SmGST3 isolated from adult S . mansoni adsorbed on alum adjuvant as described by Mishell and Shiigi (Mishell, BB, et al.. Selected Methods in Cellular Immunology, W.H. Freeman, San Francisco, pp. 1 -486, ( 1980)).
  • FIG. 5 demonstrates that mice receiving the pCMVSmGST-3 DNA responded to the treatment.
  • the mice were sacrificed 6 weeks after infection and 8 weeks after vaccination.
  • the portal vein was retrogradely perfused and the adult worms collected.
  • the livers were fixed in formalin for histological analysis and the muscle that was injected with DNA was excised and placed on dry ice for luciferase assay. Sera were collected from all mice prior to vaccination, immediately prior to infection and at sacrifice.
  • mice vaccinated with 10 ⁇ g of pCMV-GST3.7 DNA had 89% fewer eggs than mice receiving any of the other treatment regimens. This has been suggested as being the manner in which
  • mice vaccinated with DNA or isolated GST protein Eggs per unit area of mice vaccinated with DNA or isolated GST protein
  • Control 6 60.0 pRSV-Lux 5 69.7 (- 16%) p pBBssCCMMVVSSmmGGSSTT33 44 66..77 89%
  • the lymphocyte proliferation assay (26, 35) used is as follows. Briefly, spleens removed asceptically are reduced to single cell suspensions in cold RPMI 1640 medium. The suspensions are filtered through 2 layers of sterile gauze and the passing debris allowed to settle for 10 minutes. The resulting suspension is decanted and centrifuged at 900 X g for 10 minutes at 4°C and resuspended in fresh RPMI 1640. The cell concentration is determined by electronic counting (Coulter Counter) and adjusted to 10? cells/ml.
  • the cells are cultured at a density of 2 X 10 ⁇ cells/0.175 ml of RPMI 1640 containing 5% normal human, heat- inactivated serum and 20 ⁇ g of gentamicin sulfate.
  • Unstimulated control cultures receive an additional 25 ⁇ l of RPMI 1640 and stimulated cultures 25 ⁇ l of RPMI 1640 containing either 20 ⁇ g/ml Con A (2.5 ⁇ g/200 ⁇ l culture); or 24 ⁇ g GSH-agarose affinity purified 5.
  • mansoni GST about 70% of which is GST3 (3 ⁇ g/200 ⁇ l culture).
  • the supernatant is removed by aspiration and the bead pellet resuspended in 1 ml of the purified GST protein in borate buffer (pH 8.3) at 1 -2 mg/ml.
  • the protein-gel suspension is mixed overnight at 4°C on an end-over-end mixer.
  • the gel suspension is again centrifuged as above and the pellet resuspended in 3 ml of 1 M ethanolamine, pH 8.0 at room temperature to block unbound sites on the beads.
  • the beads are washed several times with sterile phosphate buffered saline (sPBS), pH 7.6, counted and adjusted to 10,000/ml.
  • sPBS sterile phosphate buffered saline
  • the ELISA procedure used is essentially as described (26) with modifications for use in detecting anti-SmGST antibodies. Briefly, the affinity purified SmGST is diluted in 0.05 M carbonate buffer at 3-5 ⁇ g/ml and 100 ⁇ l of the solution is added to each well and incubated at room temperature 1 -2 hours and then 4°C overnight. The plates are then rinsed 3 times with PBS and then nonspecific binding sites blocked with 2% bovine serum albumin diluted in PBS for 30 minutes at room temperature. The blocking solution is rinsed away by 3 washes of PBS containing 0.05% Tween 20. The plates can either be dried and stored or used as follows.
  • Each mouse serum to be tested is diluted (see below) in PBS/Tween 20 and 100 ⁇ l of this dilution added to the precoated wells (each dilution done in duplicate) and the plate incubated at room temperature 1 hr. The plate is then washed 4 times with PBS/Tween 20 and 100 ⁇ l of horseradish peroxidase-labelled goat anti-mouse immunoglobulin (Heavy and light chains combined) diluted 1 : 100 in PBS/Tween 20 added to each well and incubated for 1 hour. The plate is then washed 5 times in PBS/Tween 20 and once in PBS.
  • Each wel l receives 100 ⁇ l of the substrate solution, 0.4 mM 2,2'-azino-di(3 ethylbenzthiazoline sulfonic acid) and 2 mM H 2O 2 diluted to a final molarity in 0.05 M citrate buffer (pH 4.0). After optimal color development occurs ( 15-30 min. in the dark at room temperature) the reaction is stopped by adding 50 ⁇ l of 1.0 N
  • Spectrophotometric absorbance is determined in individual wells with an enzyme immunoassay (BioTek 308) plate reader at 405 nM with a 490 nM reference.
  • a standard curve is established by coating wells with 0.1 , 0.5, 1.0, 2.5, 5.0, and 10.0 ⁇ g of affinity-purified SmGST per well. The greatest dilution of a reference polyclonal rabbit anti-SmGST serum giving a linear recognition of the standard curve is used to determine what dilution of the test sera is examined in the ELISA assay.
  • Plasmids were prepared by standard methods that included double alkaline lysis of the bacteria propagating the plasmids, two purifications on CsCl gradients, dialysis against 1 mM Tris-HCl (pH 7.4) and 0. 1 mM EDTA, ethanol precipitation, and solvation into distilled, deionized autoclaved water as described (4- 6). Approximately 5 mg of pCMV-SmGST3 was prepared. However, should the quantity of plasmid DNA need to be replenished, the bacteria containing this plasmid is in house in a frozen stock. In addition, the pRSV-LacZ reporter gene plasmid may be used.
  • mice Each naked DNA injection has added to it 10 ⁇ g of pRSV-LacZ DNA prepared as above.
  • This control serves two purposes. First, it provides knowledge that the DNA solution for vaccination was delivered to the muscle and secondly, it indicates that foreign genes were being expressed by the muscle in the case that no immunological response to SmGST3 is observed. Additionally, a monoclonal antibody, that specifically recognizes E. coli ⁇ -gal can be used to perform an ELISA in parallel with the GST ELISA to monitor host response to the transferred genes.
  • mice are anesthetized with Metaphane until they are nonresponsive to irritative stimuli (whisker tug and paw pulling). While maintaining this level of anesthesia, a small incision through the skin perpendicular to the long axis of the quadriceps of the leg are made to expose the length of the muscle.
  • a 100 ⁇ l injection of sterile saline containi ng the desired mass of expression-grade plasmid DNA is injected into the body of the quadriceps muscle using a 1 ml tuberculin syringe and a 27 g x 0.5 in needle. The needle is withdrawn slowly so as to prevent reflux, and the incision closed with 1 -2 sterile wound clips. The mice are allowed to regain consciousness and after 1 hour are returned to the vivarium. The mice are checked daily. This procedure has been performed on 40 mice with no adverse reactions (i.e., all lived, no infections, all expressed the transferred gene).
  • T cell-mediated immunity Two measures of T cell-mediated immunity (CMI) are used.
  • An in vitro correlate of T cell clonal expansion namely the lymphocyte proliferation assay in response to specific antigen with dividing cells allowed to incorporate ⁇ pr.j jR i n t 0 newly synthesized DNA, is the primary assay.
  • mice 15 for each time period are established as follows: Negative Control Groups -- 1 ) untreated; 2) irrelevant pCMV (a plasmid with no gene inserted in the downstream polycloning site and therefore not expressing any gene product); Positive Control group — to make sure that there is one positive T cell response, this group is vaccinated with pRSV-LacZ (a plasmid encoding the E. coli ⁇ -gal actosidase enzyme) and the T cells stimulated with the commercially available enzyme.
  • pRSV-LacZ a plasmid encoding the E. coli ⁇ -gal actosidase enzyme
  • mice Three groups of mice are set up as follows: one group each is vaccinated with 10 ⁇ g, 50 ⁇ g or 100 ⁇ g of pCMV-SmGST3. Five mice from each treatment group are sacrificed on days 7, 14 and 28 after vaccination and spleen cells are removed and used in the lymphocyte proliferation assay . Lymphocytes from each animal's spleen are tested separately in a microwell assay requiring only 2.5 X 10 ⁇ cells (average spleen contains 5-7 X 10 ⁇ cells). Spleen cells are exposed to either concanavalin A (a known T cell mitogen), E. coli ⁇ -galactosidase, SmGST3, or maintained in complete medium only.
  • concanavalin A a known T cell mitogen
  • E. coli ⁇ -galactosidase SmGST3
  • the cells are labelled with 0.5 ⁇ Ci of ⁇ H-TdR (specific activity: 2 Ci/mmol) and the cells harvested 8 hours later onto glass filter strips using an automated cell harvester. The radioactivity on individual filters are measured by liquid scintillation spectroscopy. All treatment groups are setup and tested in triplicate.
  • lymphocyte proliferation assay To verify that the response seen in the lymphocyte proliferation assay is T cell-dependent, one aliquot of cells from each mouse is treated with anti-Thy 1 and complement (to destroy T cells) (35) and the remaining cells exposed to the stimulants. This treatment should abolish isotope incorporation if T cells mediate the response.
  • I I significant lymphocyte proliferation is found following vaccination with DNA encoding SmGST3, the media from the proliferation cultures is assayed using Quantikine kits from Genzyme Corporation (Cambridge, MA) specific for mouse IFN- ⁇ and IL-5 to ascertain whether vaccination elicited a Thl or
  • Th2 phenotypic response
  • the second measure of CMI used is granuloma formation around beads coated with specific antigen. This is an in vivo correlate of the histopathological response that occurs in the liver of infected individuals in response to deposition of eggs of the parasite. Groups of mice are set up in an identical fashion as described above for the lymphocyte proliferation assay but they will not be the same mice. At 7, 14 and 28 days after naked DNA vaccination, 5 ,000 Sepharose 4B beads with covalently attached SmGST3 are injected into the lateral tail vein of mice. These beads embolize in the microvasculature of the lungs and if the animal has T cells which recognize the SmGST3 on the bead's surface, granulomatous inflammation will ensue.
  • mice On days 13, 20, and 34 post vaccination (1 e., 6 days after bead embo zation) the mice are sacrificed, the lungs perfused with 10% buffered neutral formalin, and histological sections stained with hematoxy n and eosin prepared. Twenty five granulomas in each lung are measured in 2 perpendiculai p l ane s with a filar micrometer and an approximation oi the cross sectional area calculated by multiplying the two measures together (as an indication of the degree of floridness of the reaction).
  • the reaction consists of several hundred cells and the predominant types are macrophages, lymphocytes and eosinophils.
  • Granulomas are being measured in the lung because the granulomatous response is a systemic manifestation oi cell-mediated immunity and the reactions occuring in the lung around beads are more circumscribed and therefore easiei to measure and analyze than in liver.
  • This experimental pai adigm has been used in studies of T cell immunopathological mechanisms in mu ⁇ ne toxocariasis (24, 25).
  • T cell-mediated immunity has been implicated in two major aspects of the disease process.
  • the best resistance against S. mansoni has come from the use of irradiated cercana but such material is too inflammatory to use in humans.
  • dissection of the immune responses elicited by vaccination of mice with irradiated cercana indicates that the protective response is dependent on T and B lymphocyte function, but independent of complement, IgM or IgE-mediated immediate hypersensitivity (36).
  • Mice vaccinated one time with irradiated cercaria require the presence of CD4+ T cells and an antibody and macrophages.
  • the macrophages are activated by the lymphokine interferon- ⁇ (IFN- ⁇ ) which is produced by CD4+ T helper cells of the 1 st subset type ( designated Th l cells, which also secrete IL-2 and lymphotoxin).
  • IFN- ⁇ lymphokine interferon- ⁇
  • Th2 subset which produce IL-4 (mediating IgE production), IL-5 (mediating eosinophilia) and IL- 10 (which down regulates Th l cells) does not decrease resistance induced by irradiated cercaria.
  • T cells from mice immunized one time with irradiated cercaria proliferated and produ c ed I F N - ⁇ upon subsequent chal lenge with S . ma n son i antigens during in vitro culture (37) and this finding forms a basis for the use of the lymphocyte proliferation assay to detect a response to naked DNA vaccination using a plasmid encoding S. mansoni GST3.
  • SmGST3 been shown to be a protective antigen when the recombinant protein mixed with adjuvant was used to immunize mice, rats and primates against challenge infection (38). If T cell proliferation is seen, are the Thl vs Th2 cytokine assays used.
  • immune interferon gamma IFN- ⁇
  • IL-5 interleukin 5
  • Granuloma formation has been found to be a T cell- mediated process (39) and, until recently, considered to be a Thl phenomenon. This conclusion was based in part on the observation that an intradermal injection of S. mansoni egg antigens into the footpad of infected mice elicited a delayed swelling and concomitant production of IFN- ⁇ . However, administration of anti- IFN- monoclonal antibody (mAb) did not have any effect on the size or cellular composition of egg granulomas in the liver while a comparable treatment with mAb against IL-5 abrogated bone marrow, peripheral blood and granuloma eosinophils in infected mice (33).
  • mAb monoclonal antibody
  • ELISA grade microwell plates are coated with purified S. mansoni glutathione S-transferase and used in an ELISA assay. Briefly, the ELISA assay works as follows: Microwell plates are coated with specific antigen (SmGST3) and nonspecific binding sites blocked with excess milk protein. Sera to be tested are serially diluted and added to the wells. Subsequently, unbound sera is washed away, and an antibody against mouse immunoglobulin is added to detect any mouse antibodies that bound to the fixed antigen.
  • SmGST3 specific antigen
  • This latter antibody is conjugated to alkaline phosphatase and when appropriate chromogenic substrate for the enzyme is added to the well, a colored product is produced which can be quantitated at a specific wavelength using an immunoassay microplate (ELISA) reader.
  • ELISA immunoassay microplate
  • a standard curve of increasing concentrations of GST is established in 6-8 wells and a reference polyclonal rabbit serum used in place of the mouse sera.
  • SmGST3 is an invertebrate GST with immunologically different properties (speaking strictly from the amino acid sequence point of view) the human or rodent host would be expected to recognize the protein as a foreign one and therefore, SmGST3 should be immunogenic and elicit an antibody response.
  • Mice and rats which have been passively administered a mAb against SmGST3 and then challenged with infective cercaria were found to have reduced worm burdens, the female worms released fewer eggs, and the eggs that were released had poorer viability than worms recovered from mice not receiving the mAb
  • mice with different major histocompatibility complex (MHC) H-2 haplotypes are given a single dose of naked SmGST3 plasmid DNA and all the tests are conducted at the single time after vaccination for which the responses were maximal.
  • the following strai ns of mice (with their H-2 haplotypes in parentheses ) used C57BL/6 (H-2b)--this mouse strain is considered a high responder strain (42, 43); CBA (H-2k)— this strain is considered a moderate to low responder strain (42, 43); SJL (H-2s)--this strain exhibits high levels of eosinophils in response to tissue invasive helminth infections but makes virtually no IgE antibody in these infections (44); and P strain (H-2p), the only mouse strain known to not develop resistance to infection with S.
  • mice are either untreated or vaccinated with 10, 50, or 100 ⁇ g pCMV-SmGST3 DNA. At the time of maximal response mice are bled for serum and sacrificed. In those mice receiving SmGST3-coated beads, the lungs are perfused and processed for histological examination. The spleens of the remaining mice are used to establish individual lymphocyte proliferation assays stimulated with either the T cell mitogen, Con A, or the specific antigen, SmGST3. The sera collected is tested in the SmGST3 ELISA to determine relative antibody levels. Humans do not respond to antigenic stimulation as uniformly as do inbred strains of mice.
  • Baculovirus expression vector which encodes the 16.4 SmGST-3 insert is descri bed.
  • a 6X his(tidine) tag was inserted at the 5' end of the coding sequence which permits metal affinity isolation using a Ni-NTA matrix.
  • the Baculovirus Expression Vector System (GIBCO-BRL Cat. #10584-027) was used per the vendors instructions.
  • a set of custom po lymeras e chain reaction (PCR) primers were commercially synthesized to the 5' and 3' ends of the cDNA encoding the S. mansoni GST-3 contained in the pBsCMV- 16.4 plasmid.
  • the primers were designed so that an EcoR I restriction site was inserted 5' to the ATG start codon while a Hind III site was added immediately after the stop codon in the open reading frame.
  • the final construct uses an ATG start codon located 5' to a 6X his(tidine) tag and aligned in such a manner that the inserted
  • PCR product was maintained in the open reading frame.
  • the resulting 65 1 bp amplified fragment was then ligated into the pFastBac HTb shuttle vector which had been linearized by cutting with EcoRI and Hind III.
  • the ligated product was next transformed into DH5 ⁇ F' competent cells and amplified.
  • the plasmid DNA was isolated using a Qiagen Spin Prep column. Following visualization on a an agarose gel, 1 ng of plasmid DNA was used to transform DH l OBac cells. These cells contain a bacmid with a mini-attTn7 target site and a helper plasmid.
  • the mini-Tn7 element on the pFastBac plasmid can transpose to the mini-attTn7 target site on the bacmid in the presence of transposition proteins provided by the helper plasmid.
  • the transposition was carried out by incubation on LB plates containing kanamycin (50 ⁇ g/m l ; gentamicin (7 ⁇ g/ml ); tetracycline (10 ⁇ g/ml), and 100 ⁇ g/ml Bluo- gal plus 40 ⁇ g/ml IPTG. White colonies containing the recombinant bacmid were identified by blue-white screening. High molecular weight DNA was isolated by alkaline lysis from mini prep cultures.
  • bacmid DNA containing the SmGST-3 cDNA insert was used to transfcct Sf21 insect ovary cells in the presence of CellFECTIN.
  • the culture medium from this transfection was collected to use as a source of Baculovirus-SmGST-3 (Bac-SmGST-3) pending verification that the Sf21 cells were producing the S . mansoni glutathione S-transferase .
  • the cells were lysed with 5X lysis buffer (50 mM Tris, pH 7.5 ; 650 mM NaCl; 5% Triton X- 100; 50 mM NaF, 50 mM Na2HPO4; and 50 mM Na pyrophosphate).
  • the proteinase inhibitor, PMSF 100 mM stock in absolute ethanol
  • the lysis product was centrifuged at 17100 X g for 10 min at room temperature and the soluble phase separated from the pellet.
  • the pellet and supernatant were separated on a 12.5% SDS-PAGE gel and the resulting isolated proteins transblotted to nitrocellulose for immunoblotting.
  • the nitrocellulose membranes were blocked with 3 % nonfat dry milk solution, and then stained with a 1 : 160,000 dilution of a rabbit anti-S. mansoni GST-3 polyclonal antibody (provided by Dr. James Tracy, U. Wise. Vet School).
  • the membranes were washed and a goat anti-rabbit IgG conjugated to horseradish peroxidase (Sigma Chemical Co., St. Louis, MO) diluted 1 : 10.000 was applied. Following several additional washes, the bands were visualized using the ECL chemiluminescent kit from Amersham.
  • the chemiluminescent signal was collected on X-ray film and following development, the Western blot image as well as the pretransfer, Coomassie Brilliant Blue-stained replicate SDS-PAGE gel were scanned into computer storage using an HP ScanJet IICX at identical scale to facilitate alignment. The results are shown below as Figure 8.
  • the immunodetection of a strong signal at 28 kDa is proof that the Baculovirus-infected cells are indeed producing the SmGST-3 protein. It is noteworthy that little, if any, of the protein is in the pellet fraction and little, if any, is in the extracellular medium. Having verified that the cells can produce the recombinant protein, it one can purify the protein to near homogeneity by passing the lysis supernatant over a Ni-NTA column. The Ni matrix has a strong affinity for the 6X his tag which was cloned 5' to the SmGST-3 molecule.
  • Ni-NTA was purchased from Qiagen (Chatsworth, CA) and used as per the vendor's instructions.
  • the gel slurry was washed in Buffer A and incubated overnight with 1 ml of Sf21 cell extract (i.e., the resulting lysis supernatant).
  • the unbound material was washed away by pelleting the slurry and removing the supernatant (which was retained for subsequent analysis and to verify that the 28 kDa band was removed).
  • Buffer A containing 20 mM imidazole
  • adherent protein was eluted by washing the column with Buffer C containing 250 mM imidazole and 2 ml fractions collected. The presence or absence of the immunoreactive 28 kDa protein in each of the fractions was again confirmed by SDS-PAGE and chemiluminescent western blotting.
  • Figure 9 The results of one such experiment are shown in Figure 9 which suggests that the protein is being recovered in fractions 4 through 12. These fractions were next pooled, dialyzed against phosphate buffered saline and concentrated using an Amicon Centriprep with a nominal Molecular Weight Cutoff of 10 kDa.
  • Figure 10 depicts a chemiluminescent Western blot indicating that the protein was concentrated and retained immunoreactivity.
  • 100 to 200 ⁇ g of rSmGST-3 from a single T-75 flask of Sf21 cells infected at 80 to 85 confluency can be produced routinely and harvested after 3 days of infection. The resulting protein was next used to skin test mice which had been intramuscularly vaccinated 2 weeks previously.
  • One test used to evaluate the immune status of vaccinated mice is a simple skin test in which an antigen used to induce the vaccinated state is subsequently injected intradermally to see if the foreign protein is recognized.
  • the end point of the skin test is the quantification of the inflammation (here taken as increased thickness which represents the swelling due to the accumulation of an inflammatory infiltrate) present 24 hr after the skin test is begun.
  • the significance of the duration of the test over a twenty four hour period is that inflammation occurring during the first six hours is considered to be indicative of the immediate hypersensiti ve state (mediated by IgE and mast cells) while inflammation occurring at 24 hours represents the delayed hypersensiti ve state .
  • Delayed hypersensitive responses are manifestations of cell-mediated immunity and are the desired outcome of any vaccination against an infectious agent such as
  • mice vaccinated with naked DNA encoding an S. mansoni-deri ved gene manifest cell-mediated immunity groups of mice were vaccinated with either pBsCMV- 16.4 DNA (specific lest) or pBsRSV-lux (nonspecific, irrelevant DNA encoding firefly luciferase).
  • pBsCMV- 16.4 DNA specific lest
  • pBsRSV-lux nonspecific, irrelevant DNA encoding firefly luciferase
  • mice vaccinated with pBsCMV-16.4 DNA were compared to the SET values of mice vaccinated with pBsRSV-lux.
  • the results of a typical experiment are shown in the table below.
  • mice vaccinated with the pBsCMV-16.4 DNA and skin tested 14 days later have statistically significant cell mediated immunity against the SmGST-3 antigen.
  • the following references were cited herein:

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Abstract

L'invention porte sur un vaccin comportant une séquence d'ADN non infectieuse et non intégrante codant pour la glutathion-S-transférase du Schistosoma mansoni, et sur une méthode de protection contre ladite infection parasitaire.
PCT/US1997/003977 1996-03-13 1997-03-13 ADN CODANT POUR LA GLUTATHION-S-TRANSFERASE DE 28 kDa DU SCHISTOSOMA MANSONI ET SES UTILISATIONS Ceased WO1997033610A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1074612A1 (fr) * 1999-08-04 2001-02-07 Pasteur Merieux Serums Et Vaccins Utilisation de peptones végétales pour la préparation de vaccins d'ADN
US8734807B1 (en) 2013-04-06 2014-05-27 Gabriel Langlois-Rahme Preventing and curing Schistosomiasis mansoni by inhibiting Trk receptors on female Schistosoma

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5583011A (en) * 1994-07-25 1996-12-10 Board Of Trustees Of The Leland Stanford University Compositions, treatments, and diagnostics for Schistosomiasis and related diseases

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Publication number Priority date Publication date Assignee Title
US5583011A (en) * 1994-07-25 1996-12-10 Board Of Trustees Of The Leland Stanford University Compositions, treatments, and diagnostics for Schistosomiasis and related diseases

Non-Patent Citations (6)

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Title
GENE, 1993, Vol. 124, McNAIR et al., "Cloning and Characterization of the Gene Encoding the 28-kDa Glutathione S-transferase of Schistosama Mansoni", pages 245-249. *
JOURNAL IMMUNOLOGY, 1994, Vol. 153, No. 12, KHAN et al., "Construction, Expression and Immunogenecity of Multiple Tandem Copies of the Schistoma Mansoni Peptide 115-131 of the P28 Glutathione S-transferase Expressed as C-terminal Fusions to Tetanus Toxin Fragment C in a Live Aro-attenuated Vaccine Strain of Salmonella", pages 5634-5642. *
JOURNAL OF IMMUNOLOGY, 01 July 1993, Vol. 151, No. 1, RICHTER et al., "Candidate Vaccine Antigens that Stimulate the Cellular Immune Response of Mice Vaccinated with Irradiated Cercariac of Schistosoma Mansoni", pages 256-265. *
MOLECULAR AND BIOCHEMICAL PARASITOLOGY, 1990, Vol. 40, HENKLE et al., "Comparison of the Cloned Genes of the 26- and 28-Kilodalton Glutathione S-transferases of Schistosoma Japonicum and Schistosoma Mansoni", pages 23-34. *
MOLECULAR IMMUNOLOGY, 1994, Vol. 31, No. 16, PANCRE et al., "Evaluation of the Effect of Sm28GST-derived Peptides in Murine Hepatosplenic Schistosomiasis: Interest of the Lipopeptidic form of the C-terminal Peptide", pages 1247-1256. *
NATURE, 12 March 1987, Vol. 326, BALLOUL et al., "Molecular Cloning of a Protective Antigen of Schistosomes", pages 149-153. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1074612A1 (fr) * 1999-08-04 2001-02-07 Pasteur Merieux Serums Et Vaccins Utilisation de peptones végétales pour la préparation de vaccins d'ADN
US8734807B1 (en) 2013-04-06 2014-05-27 Gabriel Langlois-Rahme Preventing and curing Schistosomiasis mansoni by inhibiting Trk receptors on female Schistosoma

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