WO1997033610A1

WO1997033610A1 - DNA ENCODING 28 kDa GLUTATHIONE S-TRANSFERASE OF SCHISTOSOMA MANSONI AND USES THEREOF

Info

Publication number: WO1997033610A1
Application number: PCT/US1997/003977
Authority: WO
Inventors: Stephen G. Kayes
Original assignee: South Alabama Medical Science Foundation
Priority date: 1996-03-13
Filing date: 1997-03-13
Publication date: 1997-09-18
Also published as: AU2078697A

Abstract

A vaccine comprising a non-infectious, non-integrating DNA sequence encoding Schistosoma mansoni glutathione S-transferase and a method of protection against said parasitic infection.

Description

DNA ENCODING 28 kDa GLUTATHIONE S-TRANSFERASE OF SCHISTOSOMA MANSONI AND USES THEREOF

BACKGROUND OF THE INVENTION

Field of the Invention

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.

Description of the Related Art

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. In a significant number of infected individuals, the ensuing disease is characterized by hepatosplenomegaly, portal hypertension and esophageal varices, the latter often being fatal. Currently, 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 .

Studies have examined the effects of addition of mitomycin C-treated cells from either infected or normal mice added into lymphocytes undergoing lymphocyte proliferation (i.e., the lymphocyte transformation assay or LTA) in response to T cell mitogens (PHA and Con A) or Schistosoma mansoni egg antigens (21 -24). These studies established that unstimulated spleen cells from infected mice, but not from normal mice, induced incorporation of almost 3 times more ³H-TdR than did normal cells (22). This activity was subsequently shown to be dependent on the presence of macrophages in the cultures (23). These studies form the basis of using the lymphocyte proliferation assay as indicative of a cell-mediated immune (CMI) response in mice vaccinated against a specific schistosome protein.

Immunoregulation of pulmonary granuloma formation in toxocariasis (mice infected with the larval stage of the canine roundworm, Toxocara canis, the causative agent of human visceral larva migrans) was established. This model system correlated T cells and their subsets, which were monitored in the LTA, with the development of granulomas forming around T. cam^'s- anti gen coated CNBr-4B Sephadex beads embolized into the pulmonary microvasculature. This work forms the basis for the remaining CMI assay. A T. cams-specific ELISA to measure parasite-specific antibodies was also developed. This procedure has been modified slightly to measure anti-S. mansoni glutathione S-transferase (SmGST).

Injection of genetic constructs directly into striated muscle in vivo results in expression and, in many instances, secretion of the gene product into the circulation (4-6). Following intramuscular injection, plasmid DNA is found distributed throughout the muscle and is able to diffuse throughout muscle- associated connective tissues. Foreign transgenes have been expressed for up to 19 months but the efficiency of transfer is relatively low (2% of treated muscle) (5). Localization studies at the electron microscopic level using plasmid DNA conjugated to colloidal gold and compared to gold-tagged polyethylene glycol have shown that nucleic acids (but not the polyethylene glycol) are able to traverse the external lamina and enter myofibers through the T-tubule system, apparently associated with caveolae in the sarcolemma (7).

Since the original observations by Wolff et al. (4) that striated muscle can express injected transgenes or mRNAs, many of the variables of the technique have been clarified although many questions still remain. Of particular interest is the observation that the level of expression of transgenes tends not to be predictable although in most cases the degree of expression seems to be proportional to the amount of nucleic acid taken up. For example, a 100 gram injection into the mouse quadriceps muscle resulted in approximately 1.5 % of the fibers expressing β -gal activity as demonstrated by enzyme histochemistry 7 days after the injection. Staining could be observed up to 400 microns from the injection site. At present, the best evidence available indicates that plasmid

DNA, when taken up by skeletal muscle, remains circular and neither integrates into chromosomal DNA nor replicates (5, 6).

The expression of naked transgenes has been found to vary substantially following injection into different mice under identical conditions of injection ( 14). Subsequent studies (6) indicated that the injection technique per se is not responsible for this observed variation in transgene expression. The volume of fluid containing the genetic construct does not appear to be critical in determining uptake or expression. Likewise, neither the rate of injection (less than 30 seconds to 5 minutes) or the type of needle used to administer it (27 gauge with 0.2 cm limiting collar compared with glass capillary), seem to affect the outcome. However, conditions that lead to increased degradation of DNA can result in less uptake and expression. Interestingly, DNA dissolved in sterile saline results in 5-fold to 10-fold greater transgene activity than the same DNA dissolved in 20% sucrose. An injection of 25% sucrose 15 to 30 minutes prior to DNA injection was reported to reduce the variability in the technique (14). This presumably results from the fact that saline causes less muscle damage than the sucrose vehicle in the former case and may actually stimulate pinocytotic uptake in the latter instance. Animals which received multiple injections of the same construct over several weeks expressed less activity than did mice receiving a single injection. Moreover, naked DNA taken up by skeletal muscle tends to persist for substantial periods of time and better expression can be expected with fewer administrations. The choice of promoter sequence to drive gene transcription can influence gene expression by a factor of 1 ,000 or more ( 14). More recently, the drug Marcaine^® has been found to increase uptake and expression dramatically when given a few days prior to the DNA injection (27). The drug is slightly myotoxic and satellite cells surrounding necrotic fibers undergo mitosis which may be account for the enhanced uptake and expression.

Naked DNA techniques ( 1 1 ), and 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). Similarly, chickens were given plasmid DNA injections by one of three routes (iv, ip, or im) and subjected to an influenza virus- challenge. In these studies, 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). Cattle have also been injected intramuscularly with plasmids carrying bovine herpesvirus genes and these cattle exhibited significantly elevated titres of anti- bovine herpesvirus glycoprotein antibody and the treated animals shed significantly less virus than the control animals following infectious challenge (28) . Recently, non-human primates

(cynomolgus macaques) were injected intramuscularly with a plasmid expressing the gp l 60 gene of HIV. Animals were inoculated three times and serum collected two weeks later. This regimen led to seroconversion of all test animals and the sera was shown to contain neutralizing activity as determined by the reduction in number of microsyncytia forming in cultures of T cells exposed to HIV-I ( 1 , 13).

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.

SUMMARY OF THE INVENTION

The present invention discloses several eukaryotic expression plasmids containing genes derived from S. mansoni that serve as the basis of a polynucleotide vaccine. In this system, 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. Lastly, there is solid evidence that 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

DNA encoding a gene whose product is a good candidate for vaccine use when admixed with adjuvants and given to mice, rats, and primates. 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. Vaccination of susceptible hosts using intramuscularly injected naked DNA encoding a parasite-specific protein elicits immunological responses necessary for inducing resistance to infection and/or ameliorating the disease in such a way as to alter subsequent transmission of the disease. 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. As measures of cellular immunity, 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).

In one embodiment of the present invention, there is provided a composition of matter comprising a naked DNA vaccine for the parasitic worm, Schistosoma mansoni or human blood fluke. In another embodiment of the present invention, there is provided novel plasmids and expression vectors for use in the methods of the present invention. In yet another embodiment of the present invention, there is provided 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. Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.

Figure 1 shows a schematic drawing of the deletion of CMV promoter and replacement with α S A pro m oter . Inset=miniprep of pRcαSA cut with (S)ma I and (P)vu II.

Figure 2 shows the histochemical localization of E. coli β gal activity in mouse skeletal muscle 3 weeks after injection of 100 μl saline of b) saline with 50 μg pRSV-LacZ DNA. Orig. 400x. Figure 3 shows a photograph of a gel showing RE orientation analysis of pCMVGST3 clones. Lane 1 = Hind Ill/lambda markers , 818 and 4300 bp lanes are correct orientation.

Figure 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 μ g pSmGST3, 50 μg pSmGST3, or a subcutaneous injection of 25 μg 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. Note the over expressed 28 kDa band in lane 2A and the recovery of a discrete band at 28 kDa in lane 4A (arrow) which is completely absent in lane 4B. Gel was composed of 12% polyacrylamide and was stained with coomassie blue, destained, dried and scanned with a HP Scanjet. 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. A 12.5% SDS-PAGE was run as follows: Lane M = molecular weight Markers (in kDa) indicated at left of image; Lane U = uninfected Sf21 cell lysate; Lanes 3, 4, and 5 = Sf21 cell lysates harvested at 3, 4 or 5 days after infection with Bac-SmGST-3. Note the absence of the 28 kDa band in lane U and its over expression in lanes 3, 4, and 5. (Figure 8B) The gel in Figure 8A was electroblotted to a nitrocellulose membrane and immunostained with polyclonal rabbit anti-S. mansoni GST-3 followed by staining with goat anti-rabbit IgG conjugated to horseradish peroxidase. Immunolocalization was observed using an ECL kit from Amersham followed by image capture on X-ray film. Both Figures 8A and Figure 8B are computer scans of the original images. Note the strong band at 28 kDa indicative of the production of SmGST-3. The faint images below the band are degradation products of the 28 kDa protein.

Figure 9 shows a SDS-PAGE of various stages during the isolation procedure of recombinant SmGST-3 protein from Bac-

SmGST-3 infected Sf21 cell lysates. Lanes contain the following: W = whole unfractionated Bac-SmGST-3 infected Sf21 cell-lysate; A = same lysate after adsorption with Ni-NTA slurry (en batch); 1 , 2, and 3 = washes with Buffer A or B to remove weakly adherent proteins; MR = 10 kDa molecular weight markers; Fractions 1 - 4 =

2 ml fractions were collected while eluting the Ni-NTA slurry with Buffer C containing 250 mM imidazole. Note the presence of the 28 kDa bands in fractions 4 through 12.

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

Figure 9. The image represents lanes loaded with the following:

Lane 1 = whole lysate (compare with Lane W in Figure 9); Lane 2 = post adsorption fraction showing depletion of immunoreactive band; Lane 3 = 10 kDa ladder (position determined by comparison with SDS-PAGE used for blotting); Lane 4 = filtrate from 10 kDa cutoff Centriprep concentrator indicating that 28 kDa protein was partitioned out of this solution; Lane 5 = retentate from 10 kDa cutoff Centriprep concentrator indicating that 28 kDa protein was partitioned into this fraction.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a vaccine comprising a non-infectiou s , non-inte grating DNA sequence encoding Schistosoma mansoni glutathione S-transferase. Preferably, the DNA comprises a plasmid encoding a protein, polypeptide or peptide which is operably linked to a promoter. In one embodiment, the plasmid is pCMVSmGST-3. In other embodiments, 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 . Preferably, 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. Generally, a person having ordinary skill in this art would be able to determine optimal dosage of the compositions of the present invention to achieve the desired outcome. Generally, the plasmid is administered in an animal in a dose of from about 25 μg to about 200 μg. Preferably, the plasmid is administered into muscle although it is possible it could be adminstered into another site in the body. Generally, 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. Generally, a person having ordinary skill in this art would be able to determine optimal dosage of the compositions of the present invention to achieve the desired outcome. Generally, the plasmid is administered in an animal in a dose of from about 25 μg to about 200 μ g. Preferably, the plasmid is administered into muscle although it is possible it could be adminstered into another site in the body. Generally, the plasmid would be administered between 1 and 6 times.

In the present invention, 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. At weekly intervals muscles were removed, frozen sections prepared, the sections stained for β-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 ). Next, 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.

To establish that this construct was both non-toxic to mammalian cells and to verify that cells transfected with pCMV- SmGST-3 expressed the GST gene, 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). Finally, 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. Figure 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 (GST) (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. 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).

All 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). In 1988, 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). It has been established that a) 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.

GSTs are good candidates for use in vaccines against S. mansoni (29-32). In the rodent model of S. mansoni infection a Mr 28,000 molecule (Sm28 or P28) was identified as a major surface protein (where it may mediate the conjugation of GSH to antischistosomal drugs) on larval schistosomes (schistosomules) and was subsequently identified as a GST (32). From a routine screening of S. mansoni RNA translation products using immune serum from infected mice, a protein was identified which could be detected on the surface of the immature S. mansoni worm and the adult. 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).

Determination of whether vaccination with a polynucleotide (i.e. naked DNA) will yield a better immunization than the more conventional vaccination using a pathogen-derived protein with an adjuvant such as aluminum potassium sulfate (alum, which is acceptable for human and veterinary use) is desirable. The genetic constructs were made and several milligrams of expression-grade DNA were stored at -80°C. To demonstrate that cellular immunity in murine schistosomiasis is induced, lymphocyte proliferation to specific S. mansoni antigen (SmGST-3), an already documented manifestation of cellular immunity and pulmonary bead granuloma formation (34) are used. Likewise, the development of mouse anti-SmGST antibodies (i.e., the humoral immune response) are monitored by ELISA.

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.

EXAMPLE 1 Modification of a eukaryotic expression vector

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. 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 α -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. First, 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. From a stock of custom fragments, a 6.2 Kb pαSK fragment of genomic DNA was obtained carrying the chicken a -skeletal actin (α 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 αSA fragment isolated from a 1 % agarose gel. The 5 Kb pRc plasmid and the 675 bp αSA fragment were ligated using T4 ligase. The ligation products were then transfected into TOP10F' and 22 colonies were screened for plasmid DNA. Because blunt end ligations can result in the fragment being ligated in either direction, plasmids were cut with Sma I which cuts asymmetrically one time within the αSA sequence and once within the remaining pRc sequence. If the α 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 pRcαSAδ 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 pRcα 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 α SA promoter sequence with the promoter sequence inserted in the 3'-->5' orientation was also stocked (pRcαSA 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 pRcASKδ, 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.

EXAMPLE 2 In vivo gene transfer of pBsRSV-βGal and visualization of gene expression

In order to verify that transferred genes are in the intended tissues and that the foreign genes are being expressed, a eukaryotic reporter plasmid in which the E. coli β-galactosidase (β- gal) gene is inserted into the polycloning site can be used to verify both properties. By staining frozen sections for β-gal activity, fibers in which the transgene are being expressed will stain positively while all other fibers fail to stain. Groups of mice were injected in the quadriceps muscle with either saline or 50 μg of purified pBsRSV-βGal DNA. Mice were sacrificed after 1 , 2, or 3 weeks and the injected muscles removed, snap frozen in methyl butanol on dry ice, and 10 μm frozen sections prepared. The sections were stained with X-gal in the presence of potassium ferriferrocyanide. All muscles injected with the pBsRSV-βGal plasmid had scattered myofibers which stained intensely blue- black indicating that the β-Gal gene product, β-galactosidase, was being expressed. None of the saline-injected muscles exhibited positive staining (Figure 2).

EXAMPLE 3

Fabrication of pBsCMV-GST3

A plasmid, pGT16.4, carrying the cDNA encoding the 28 kDa glutathione S-transferase (= SmGST3) situated between two Eco RI sites was obtained from Dr. Jim Tracy (University of Wisconsin Veterinary School). 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 .

EXAMPLE 4

Isolation of recombinant SmGST3 protein by GSH-agarose affinity chromatography

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).

EXAMPLE 5

Transfer of pBsCMV-GST3.7 in to HEK 292 cells

To establish that this construct was both non-toxic to mammalian cells and to verify that cells transfected with pBsCMV- GST3.7 expressed the GST gene, HEK 293 cells in tissue culture were transfected by the CaPθ4 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. Soluble extracts of the monolayers over expressed GST activity when using chloro-dinitrobenzene (CDNB) as the electrophilic substrate in the assay described by Habig and Jakoby (34) as compared to cells transfected with an irrelevant plasmid (See Figure 4). This demonstrated that the construct was not toxic and suggested that the plasmid could be used in a gene transfer protocol for live animal experiments.

EXAMPLE 6

Addition of Xba I restriction sites to either end of SmGST3

To make it easier to work with the cDNA encoding SmGST3, (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.

EXAMPLE 7

Vaccinate- and-challenge experiment

BALB/c mice were injected intramuscularly (im) (vaccinated) with 10 or 50 μ 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 μg 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)). Results are shown in Figure 5 and were based on the splenic mass increase which correlates with lymphocyte proliferation in the white pulp. Figure 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. To confirm that plasmid DNA was retained and expressed in skeletal muscle for the 8 week duration of the experiment, muscles that had been removed at necropsy and snap frozen were subjected to the luciferase assay. The data from this assay are shown in TABLE I, and based on the pRSV-Lux group, indicate that DNA injected into these mice persisted for the duration of the experiment. Worm burdens did not appear to be affected by polynucleotide vaccination (Table II).

TABLE I

Luciferase luminometry on skeletal muscle from mice injected with either pRSV-Lux or pBsCMV-GST3.7 DNA eight weeks previously

TREATMENT n Lux units: mean±S E

Lux Std 1 unit 1 1 0,75 8

Lux Std 5 unit 1 57 ,765

None-Inf. only 4 484±99 pRSV-Lux 10 μg 5 53 ,952±25 , 889 pSmGST3 10 μg 4 833±308 pSmGST3 50 μg 4 468 ±42 TABLE II

Portal perfusion of mice 6.5 weeks after injection with 200 S. mansoni cercariae

Recoveries Control pRSV-Lux pBsCMVGST pBaCMVGST SmGST+Alum

10 μg 50 μg

Males 4 0 50 26 65 90

Females 4 0 72 26 69 67

Total 8 0 122 52 135 147

(n) 6 5 4 5 5

WWoorrmmss// 1133..33 24.4 13 27 29.4

Mouse

The luciferase data at 8 weeks after DNA injection into these mice demonstrate that the injected DNA did persist for the duration of the experiment consistent with the findings of others. However, worm burdens did not appear to be affected by polynucleotide vaccination. However, when the number of S . mansoni eggs per unit area of liver was examined in H & E stained sections, mice vaccinated with 10 μg 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

SmGST-induced vaccination manifests itself. It is worth pointing out that the number of worms per mouse in the control group and the pCMV-GST3 group in Table II is almost identical indicating that different numbers of worms can not account for the reduced egg counts in the SmGST3 group. This is compelling evidence that naked DNA vaccination is an effective modality to control schistosomiasis. A decrease in the number of eggs deposited per female results in less morbidity per patient (less liver involvement) and, more importantly, fewer eggs to reach the environment and spread the infection to new snails. TABLE III

Eggs per unit area of mice vaccinated with DNA or isolated GST protein

Treatmen t n Eggs/100 mm2. liver % Reduction

Control 6 60.0 pRSV-Lux 5 69.7 (- 16%) p pBBssCCMMVVSSmmGGSSTT33 44 66..77 89%

SmGST + Alum 5 46.0 23%

Table III: Eggs pi εr unit are f liver of mice vaccinated with DNA or isolated GST protein. Eggs (25/mo.) counted in H&E stained, 5 μm sections taken at 125 μm intervals. n=number of mice.

EXAMPLE 8

Lymphocyte Proliferation Assay

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 μg of gentamicin sulfate. Unstimulated control cultures receive an additional 25 μl of RPMI 1640 and stimulated cultures 25 μl of RPMI 1640 containing either 20 μg/ml Con A (2.5 μ g/200 μ l culture); or 24 μ g GSH-agarose affinity purified 5. mansoni GST (about 70% of which is GST3) (3 μg/200 μl culture). Cultures are incubated in 96 well flat bottom plates with loose-fitting covers in a 37°C incubator gassed with 5% CO2 in air with saturated humidity. On the morning of the 5th day (4.67 days of culture) 0.5 μCi of tritiated thymidine (^H-TdR, Sp. Act. = 2.0 Ci/mmol) is added in 25 μ l of RPMI 1640 and the cultures incubated an additional 8 hours. The cells are then harvested onto glass fiber filter strips using an automated cell harvester (BRANDEL M24V-Special). The amount of isotope incorporated into nascent DNA is determined by liquid scintillation spectroscopy after immersing the individual filters into vials of Econofluor LSC cocktail. All LSC data is corrected for quenching and expressed as disintegrations per minute (DPM).

EXAMPLE 9

Bead Granuloma Formation

The method for studying bead granuloma formation was used exactly as described previously (24, 25) except that GSH- agarose affinity-purified SmGST is substituted for methylated bovine serum albumin or T. canis antigens. Briefly, freeze-dried CNBr-activated Sepharose 4B beads (0.5 g) will be hydrated for 15 minutes in 100 ml o 1 mM HC1, sized using 41 and 52 μm Spectromesh macro filtration screens to achieve a uniform bead population (46 ± 8 μ m ) which is then concentrated by centrifugation at 1 ,000 X g (4 times) for 10 minutes. 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. Intravenous injection of 0.5 ml of the bead suspension via the lateral tail vein of mice anesthetized with Metaphane initiates bead granuloma formation. Six days later the mice are sacrificed, the lungs perfused with 2.5 ml of 10% buffered neutral formalin, and histological sections cut and stained with H & E. Granulomata forming around the beads are quantitated by planimetry . EXAMPLE 10

ELISA

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 μg/ml and 100 μl 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 μ 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 μl 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 μl 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 μl of 1.0 N

NaF to each well. 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 μg 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.

EXAMPLE 11

Plasmid Preparation and Naked DNA Vaccination

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. Each naked DNA injection has added to it 10 μg of pRSV-LacZ DNA prepared as above. At the time the mice are sacrificed, the quadriceps muscle that was injected, as well as the contralateral quadriceps i s dissected out, snap frozen in methyl butanol on dry ice, and sectioned on a cryostat for demonstration of the bacterial enzyme, β -galactosidase (2% X-Gal in potassium ferriferrocyanide). 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 β-gal can be used to perform an ELISA in parallel with the GST ELISA to monitor host response to the transferred genes.

To perform the naked DNA vaccination, 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 μl 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). EXAMPLE 12

Analysis of Data

All data deri ved from experiments containing multiple groups are analyzed by multivariate analysis of variance (MANOVA) with post hoc testing (Duncan's multiple range test or Student-Newman-Keuls multiple range test). Significance is assessed at the p < 0.05 level of confidence in all cases. All data are analyzed using the NWA Statpak software (Northwest Analytical, Inc . , Portland, OR) running on a laboratory PC. Comparisons between two groups only are performed using the Student's T test.

EXAMPLE 12

The detailed sequences of the plasmids used herein are provided in Figures 7 A & 7B . SmGST3 plasmids in their bacterial host have been frozen down as glycerol stocks.

EXAMPLE 13

Development of cellular immune responses of mice vaccinated with naked DNA encodi n g Sch istosoma manson i glutathione S- tran sferase 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_nt₀ newly synthesized DNA, is the primary assay. To demonstrate that naked DNA vaccination stimulates a T cell-mediated immune response groups of 15 mice (5 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 β -gal actosidase enzyme) and the T cells stimulated with the commercially available enzyme. Three groups of mice are set up as follows: one group each is vaccinated with 10 μg, 50 μg or 100 μg 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 β-galactosidase, SmGST3, or maintained in complete medium only. After 4.67 days of culture, the cells are labelled with 0.5 μCi 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.

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-γ 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. 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). To account for sections through the edges of the bead, the cross sectional area of the bead are subtracted from the areas of the granulomas Using granuloma area of the untreated mice harboring SmGST3 coated beads as the denominator, a Granuloma Index for all other groups of mice are calculated using the formula GI = ( [Test Grp-Untreated Grp]/Untreated Grp) Besides measuring the approximate area of the granulomatous l eaction, the types of cells found in the reaction are noted. If the animal does not recognize the SmGST3 on the beads, a foreign body reaction occurs in which the beads are surrounded by only a few cells most of which are fibroblasts. If the SmGST3 antigen is recognized, 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 muπne toxocariasis (24, 25).

In schistosomiasis mansoni, T cell-mediated immunity has been implicated in two major aspects of the disease process. The mechanism of protection against infection following immunization by pπoi exposure to parasite antιgen(s) and the granulomatous response to eggs which accumulate in the host tissues both have been shown to be T cell-dependent reactions. To date the best resistance against S. mansoni has come from the use of irradiated cercana but such material is too inflammatory to use in humans. Howevei , 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-γ (IFN-γ) 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). Depletion of cells of the 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 -γ 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. Because naked DNA vaccination has been used to vaccinate laboratory and agricultural animals against several viral i nfections where Th l -mediated immunity is absolutely essential , immune interferon gamma (IFN-γ) as indicative of a Th l response and interleukin 5 (IL-5 ) as representative of a Th2-mediated response is quantified. While it is expected that the naked DNA vaccination will give a Thl response, knowing whether or not it does, permits a modification of the vaccine protocol wherein a plasmid encoding IL-12 which drives ThO cells to become Thl cells, can be incorporated into the antigen-encoding DNA to yield a multigenic vaccine.

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-γ. 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). These experiments were done on acutely infected mice at a time when the granulomatous response is maximal, and so too, are eosinophilia and IgE levels. These later responses depend on Th2 lymphokines (33). This suggests that Th2, and not Thl , cells are mediating granuloma formation in acute schistosomiasis. From the perspective of vaccine development, granuloma formation represents a quantifiable aspect of host response to the parasite (i.e, number of eggs per gram liver). If vaccination can reduce the number of eggs released per female worm (as measured by the number of granulomas around eggs in the livers) then the vaccinated population would be putting out fewer eggs into the environment and this should have a major effect on the subsequent transmission of the disease.

EXAMPLE 14

Development of humoral immune responses of mice vaccinated with naked DNA encoding Schistosoma mansoni glutathione S- transferase

In order to measure anti-SmGST3 antibody, peripheral blood sera are collected from each mouse prior to being randomized into a treatment group and then again immediately prior to being infected. To determine whether mice are making anti-SmGST3 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. 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. To make this assay semiquantitative, 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. Thus, the present invention determines if vaccination elicits a specific antibody response and the relationship of this response to the amount of DNA administered and the length of time after administration.

Because 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

(41). These properties of a vaccine would go a long way towards disrupting the transmission of this disease by reducing the infective egg burden released into the water. This in turn would protect the snail population from participating in the obligatory asexual phase of the life cycle. Additionally, it is the egg deposited in the liver that elicits the granulomatous response and it is this very response, which serves to sequester S. mansoni- d eτi v ed hepatocellular toxins, that ultimately destroys the liver, and generates the portal hypertension and potentially lethal esophageal varices. Fewer eggs would ameliorate this aspect of the infection. These are properties that increase the value of targeting S. mansoni GST with a vaccine. Also, the presence of anti-SmGST3 antibodies in the serum validates the expression of the polynucleotide injected and gives a clinical basis for knowing a priori whether a person was in fact, immunized.

EXAMPLE 15

Effect of the H-2 haplotype on immune responses of mice vaccinated with naked DNA encoding S. mansoni glutathione S- transferase

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. mansoni (45). Mice are either untreated or vaccinated with 10, 50, or 100 μg 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. While most inbred strains that have been studied respond to vaccination with irradiated cercaria by developing moderate to high levels of protective immunity against a challenge infection, the P strain always fails to do so following vaccination (45). Peak levels of IgG to larval schistosome surface antigens produced by P strain mice are equivalent to those in high responder strains of mice and are of the same isotypes which suggests that antibody titers alone are not what determines protective immunity. However, antibody against SmGST3 may decrease transmission by reducing fecundity and egg viability.

EXAMPLE 16

Production of Baculovirus Expression Vector for Production of Recombinant SmGST-3 Protein in Insect Cells

In order to determine whether a vaccination "takes" and to evaluate the relative strength of the immune responses of animals vaccinated with polynucleotides, ample quantity of the protein which is encoded by the DNA used to vaccinate was necessary. Isolation of the natural protein from the parasite itself is tedious, time consuming, expensive, and difficult. The use of gene transfer technology can obviate this difficulty by inserting the gene for the protein into a virus which can infect insect cells that can be maintained in tissue culture. Such cells can be lysed and the protein easily purified. The spent culture medium can be used as a source of new virus to infect subsequent cultures of insect cells and thus, an unlimited quantity of recombinant protein can be obtained for use in immunoassays. The production of a Baculovirus expression vector which encodes the 16.4 SmGST-3 insert is descri bed. To facilitate the purification of the recombinant protein, 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.

EXAMPLE 17

Construction of A Baculovirus expressing the SmGST-3 ( 16.41 cDNA

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 α 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 μ g/m l ; gentamicin (7 μg/ml ); tetracycline (10 μg/ml), and 100 μg/ml Bluo- gal plus 40 μg/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. Finally, 5 μg of 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 .

EXAMPLE 18

Screening of Sf2 1 Cells for Production of SmGST-3 Recombinant Protein frSmGST-3 )

At either 3, 4, or 5 days after infecting Sf21 cells with Bac-SmGST-3, 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), was added to the lysis buffer i mmediately prior to use (5 mM final concentration). 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.

EXAMPLE 19

Purification of 6X-his tagged rSmGST-3 Using Ni-NTA Metal Affinity Chromatography

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). Next 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). Following several washes in Buffer A (containing 20 mM imidazole) to remove weakly binding products from the gel, the slurry was transferred to a mini column and washed again, in Buffer B followed by another wash with Buffer A. Finally, the 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.

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. Now 100 to 200 μg 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.

EXAMPLE 20

Skin Testing of Mice Vaccinated with pBsCMV- 16.4 DNA Using the Recombinant GST Protein

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

Mycobacterium tuberculosis or S. mansoni.

To evaluate whether 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). Fourteen days after vaccination the thickness of the outer ear was measured with a precision pocket micrometer (Mi tutoyo Instruments #7309) just prior to the intradermal injection of approximately 30 μl of 100 μg/ml of recombinant SmGST3 protein in buffered saline. The ear was measured again at twenty four hours and specific ear thickening (SET) in micrometers was calculated as: SET = (Thickness at 24 hr minus Thickness at 0 hr)

To interpret the data, the SET values of 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.

Vaccine DNA no of mice SET Value±SD p value*

50 μg 16 3 73.3 ± 5.8 < 0.05

100 μg 16.4 3 76.7 ± 15.3 <0.05 lux 3 46.7 ± 15.3

* single tail Students t test comparing 16.4 DNA vs. lux value

These results indicate that 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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Claims

WHAT IS CLAIMED IS:

1. A vaccine comprising a non-infectious, non- integrating DNA sequence encoding Schistosoma mansoni glutathione S-transferase.

2. The DNA sequence of claim 1, wherein said DNA comprises a plasmid encoding a protein, polypeptide or peptide which is operably linked to a promoter.

3. The DNA sequence of claim 1, wherein said plasmid is pCMVSmGST-3.

4. The DNA sequence of claim 1, wherein said plasmid is selected from the group consisting of pBsCMV-16.4 DNA and pRc/ASK8-SmGST3.

5. 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 Schistosoma mansoni glutathione S-transferase.

6. The method of claim 5, wherein said vaccine composition comprises a plasmid encoding a protein, polypeptide or peptide which is operably linked to a promoter.

7. The method of claim 6, wherein said plasmid is pCMVSmGST-3.

8. The method of claim 6, wherein said plasmid is selected from the group consisting of pBsCMV-16.4 DNA and pRc/ASK8-SmGST3.

9. The method of claim 6, wherein said plasmid is administered in an animal in a dose of from about 25 μg to about 200 μg .

1 0. The method of claim 6, wherein said plasmid is administered into muscle.

1 1 . The method of claim 6, wherein said plasmid is administered between 1 and 6 times.

1 2. 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 the plasmid of claim 6.

1 3. The method of claim 12, wherein said plasmid is pCMVSmGST-3.

14. The method of claim 12, wherein said plasmid is pBsCMV- 16.4 DNA.

1 5 . The method of claim 12, wherein said plasmid is administered in an animal in a dose of from about 25 μg to about 200 μg .

1 6. The method of claim 12, wherein said plasmid is administered into m uscle.

1 7. The method of claim 12, wherein said plasmid is administered between 1 and 6 times.