IMMUNOGENIC POLYPEPTIDE AND METHOD FOR PURIFYING IT

Abstract: This invention relates to a synthetic polypeptide (preferably produced by recombinant DNA techniques) comprising an amino acid sequence incorporating (a) a portion of the P.vivax circumsporozoite (CS) protein including the region of the repeat immunodominant epitope of said protein and (b) another portion of such protein which is conserved among the different malarial species; and to a method for purifying such a polypeptide.

Description: IMMUNOGENIC POLYPEPTIDE AND METHOD FOR PURIFYING IT This patent application is a continuation-in-part ap plication of copending U.S. patent application Serial No. 754,645 filed on July 9, 1985 in the names of Arnot, D.E. e al, and co-pending PCT application No. US86/01373 filed on Jun 24, 1986 both assigned to New York University. Both of thes applications are incorporated by reference herein. Field of the Invention This invention relates to an immunogenic polypeptid expressed by a recombinant yeast and- comprising an amino aci sequence incorporating a portion of the P.vivax cir cumsporozoite (CS) protein including the region of the repea immunodominant epitope of said protein; and to a method fo purifying this polypeptide. The immunogenic properties of the P.vivax CS protei and, in particular, those of a subsequence of this protein hav been previously described, in the above-identified Arnot et a application and in Arnot, D.E. et al. Science 2_3_0:815-818, 1985 (also incorporated by reference) . In fact, the entire P.vivax CS gene has been identifie and its sequence described in the above-mentioned documents. As described therein, the P.vivax CS protein comprises a central region of 19 tandem repeats of the sequence: Asp-Arg-Ala-Asp/Ala-Gly-Gln-Pro-Ala-Gly or, by another system of notation: D R A D/A G Q P A G This region contains the repeat immunodominant epitop of the P.vivax CS protein. (The correspondence between the two notation systems i as follows: A - alanine, C - cysteine, D « aspartic acid, E glutamic acid, F = phenylalanine, G «= glycine, H « histidine, = isoleucine, K = lysine, L «* leucine, M = aethionine, N asparagine, P « proline, Q ■» glutamine, R = arginine, S serine, T « threonine, V * valine, « tryptophan, and Y tyrosine.) Synthetic peptides consisting essentially of 18 ami acid residues (i.e. two repeats of the above repeating sequen and cyclic permutations thereof — such as Asp-Gly-Gln-Pro-Al Gly-Asp-Arg-Ala) are recognized by antibodies to the nati P.vivax CS protein and in turn generate antibodies (wh injected in mammals) which recognize, and bind to, the nati CS protein. Hence, such peptides have utility in a vacci against malaria. One possible approach to making a vaccine again P.vivax malaria would be to synthesize immunogenic peptides i eluding a sequence corresponding to that of the immunodomina epitope of the P.vivax CS ' protein (i.e., containing one a preferably two or more repeats) . In the case of P. ivax. ho ever, because the sequence of the repeating unit of the protein is rather long (in contrast to that of P.falciparu classical peptide synthesis cannot be reliably used. Use classical synthetic techniques to make longer peptides is esp cially inconvenient when practiced on a large scale, as wou be necessary for manufacture of a malaria vaccine that would distributed to millions of people worldwide. Moreover, coupling of the synthesized peptide to larger molecule that would play the role of a carrier adjuvant would be necessary. In addition, in most instance only a minor proportion of antibodies to a synthetic pepti recognize the same sequence in the native protein. That i even if the synthetic peptide coupled to a carrier protein shown to be immunogenic, most of the antibodies produced m not mediate protective immunity against the pathogen. In t respect, the synthetic peptide (NANP)3 which is a candidate preparing a vaccine against P.falciparum is exceptional, si at least 70% of the antipeptide antibodies recognize malaria sporozoites. The explanation for this unusual find is probably that the (NANP)3 peptide contains many prolines and asparagines (N) , amino acids which are frequently found reverse turns of the protein molecule. Perhaps in t instance, a preferred configuration of (NANP)3 in soluti mimics that of the same sequence in the native CS prote However, from the examination of the amino acid sequence of P.vivax repeats, it does not seem likely that a P.vivax pept will behave similarly to (NANP)3. For these reasons, an alternative technique was sou for manufacturing an immunogenic peptide that could be used a vaccine against P.vivax malaria. The present invent looked to recombinant DNA and genetic engineering techniques express immunogenic polypeptides that would be used to con immunity to mammals against P.vivax malaria. Although the technology was readily available for co tructing recombinant bacteria that would express a portion all of the repeating amino acid sequence of the P.vi protein, the present inventors searched for an alternat expression system for the following reasons: First, the expression system should be reliable able to produce the polypeptide of interest consistently with a high yield. Bacteria can be difficult to handle w produced in mass culture and overexpressing a foreign prot product. Second, and more important, expression products bacteria are often difficult to purify from pyrogenic purities and other inflammatory and toxic agents that eit are co-expressed by the bacteria, or are necessary additives a bacterial growth medium. Third, expression products of bacteria are most of fusion proteins, that is, they contain additional non-relev sequences originating from the genes associated with the b teria. The present inventors looked to yeast expression sy tems, which have been substantially improved by recent advanc in the field of recombinant DNA technology. Yeasts are hardi organisms than bacteria and much easier to grow in ma culture. Moreover, recent advances in yeast genetics a cloning have increased the yields of yeast expression systems. Purification of expression products of recombina yeast systems is not a priori more complicated than purific tion of products of bacterial recombinant systems and depen mostly on the characteristics of the particular protein soug to be purified. Nevertheless, such purification is general more complicated than that of a peptide or protein produced classical peptide synthetic techniques. The present inventors chose a general yeast expressi system that is the subject matter of United States Pate Application Serial No. 868,639 filed on May 29, 1986 in t name of R.L. Burke et al and assigned to Chiron Corporatio This application is incorporated by reference herein. portion of the P.vivax gene was chosen (for incorporation in the yeast organisms) which included the entire taπdem repeated sequence plus another segment preceding the repe region (when the sequence is read from the N-terminal to the terminal) . This segment incorporates a sequence that is high conserved in all malarial species and has been previously fou to be immunogenic in its own right. (See U.S. Patent Applic tion Serial No. 649,903 of V. Nussenzweig et al filed September 18, 1984 assigned to New York University and inco porated by reference herein.) Objects of the Invention Accordingly, it is an object of the present inventi to provide an immunogenic yeast-engineered polypeptide immun chemically reactive with monoclonal antibodies against P.viv circumsporozoite (CS) protein, and useful in a vaccine prepar tion against malaria. It is another object of the present invention to pr vide a method for making the foregoing immunogenic polypepti which could be practiced on a large scale and with a yield. Another object is to provide an immunogenic polypep suitable for incorporation in an anti-malaria vaccine prep tion. Another object is to provide an immunogenic polypep suitable for use in an anti-malaria vaccine preparation wit being coupled to a carrier. Another object is to provide a method for purifying foregoing yeast-engineered polypeptide from an impure prep tion thereof comprising lysed yeast cell material and y culture media. These and other objects of the invention will be a rent to those skilled in the art in view of the pre specification, accompanying claims and appended drawings. Brief Description of the Drawings Figure 1 is a diagram of the construction of the y expression vector used in the present invention. Figure 2 is a map of yeast plas id pAB24. Figure 3 is a polyacrylamide gel obtained using lys from P. vivax/pAB2 -transformed yeast and control yeast. Figure 4 is a Western analysis of lysates from ye transformed with the P. vivax/pAB2 vector. Figure 5 is a plot of (a) the optical density prof of the material purified according to the invention (so line) ; (b) the conductivity of the eluting buffer (solid li black points) ; and (c) the percent activity of eluted fr tions in inhibiting the binding of antibody to native protein. Figure 6 is a radioautograph of a SDS-PAGE gel dem strating the purity of the engineered polypeptide when made purified in accordance with the present invention. Figure 7 is a radioautograph of an isoelectric focus gel showing the engineered polypeptide of the present inv tion. Figure 8 is a standard curve for a competitive rad immunoassay and shows the inhibition of binding of labeled, gineered CS polypeptide to anti-vivax CS protein antibodies b the presence of unlabeled engineered polypeptide of the prese invention. Figure 9 is a plot of the amount of radiolabeled goa anti-mouse IgG recognizing mouse antisera bound to engineer CS polypeptide, as a function of the mouse serum dilution. Figure 10 is a plot of the binding of labeled mou antisera (raised against the engineered peptide) to immobiliz synthetic 18-amino acid (repeat) vivax CS peptide, as function of the antibody concentration. Figure 11 is a plot of the inhibition by synthet vivax CS repeat peptide of the binding of mouse antisera immobilized engineered CS polypeptide prepared and purifi according to the present invention, as a function of t concentration of the synthetic (inhibiting) peptide. Figure 12 is a plot of the binding of mouse antisera immobilized synthetic non-repeat peptide, as a function of t antisera dilution. Summary of the Invention This invention relates to a yeast-engineered polype tide having an amino acid sequence consisting essentially the amino acid sequence: A E P K N P R E N K L K Q P G D R A D G Q P A G D R A D G Q P A G D R A D G Q P A G D RA A G Q P A G D R A D G Q P A G D R A D G Q P A G D R A D G Q P A G D R A D G Q P A G D R A A G Q P A G D R A A G Q P A G D R A D G Q P A G D R A A G Q P A G D R A D G Q P A G D R A A G Q P A G D R A D G Q P A G D R A A G Q P A G D A A G Q P A G D R A A G Q P A G D R A A G Q P A G N G A G G Q A A G G N A G G Q G Q N N E G A N A P N E K S V K E Y L D KV R A T V G T E W T P and produced by a method comprising the steps of: (a) obtaining a 15 kilobase restriction endonuclea Bgl II P. vivax CS DNA fragment in a suitable vector; (b) subcloning said fragment in a suitable vector; (c) obtaining a 4.1 kilobase P. vivax DNA fragme coding for said amino acid sequence; (d) incorporating said fragment into a yeast expre sion vector, (e) transforming yeast with said expression vector, (f) culturing said yeast under conditions which al expression of the protein encoded by said vector, an (g) harvesting the medium containing said expres protein. Detailed Description of the Invention The P.vivax CS gene is set forth below (together w the amino acid sequence for which it codes) [N- terminus] ATGAAGAACTTΛTTCTCTTGGCTGTTT raCCATCCTGTTGGTGGACT M K N F I L L A V S S I L L V D TTCCCCACGCACTGCGGGCACAATGTAGATCTGTCCAAGGCCATAAACTTAAATGGAGTAAA F P T H C G H N V D L S K A I N L N G V N TTCAATAATGTAGACGCCAGTTCACTTGGCGCGGCACACGTAGGACAAAGTGCTAGCCGAGG F N N V D A S S L G A A H V G Q S A S R G AGGCCTTGGCGAGAACCCAGATGACGAGGAAGGAGATGCTAAAAAAAAAAAGGATGGAAAGA G L G E N P D D E E G D A K K K K D G K GCAGAACCAAAAAATCCACGTGAAAATAAGCTGAAGCAACCAGGAGACAGAGCAGATGGACA A E P K N P R E N K L K Q P G D R A D G Q CAGCAGGAGACAGAGCAGATGGACAGCCAGCAGGTGATAGAGCAGATGGACAACCAGCAGGA P A G D R A D G Q P A G D R A D G Q P A G TAGAGCAGCTGGACAACCAGCAGGAGATAGAGCAGATGGACAGCCAGCAGGAGACAGAGCAG R A A G Q P A G D R A D G Q P A G D R A GGACAGCCAGCAGGAGACAGAGCAGATGGACAACCAGCAGGAGACAGAGCAGATGGACAACC G Q P A G D R A D G Q P A G D R A D G Q P CAGGTGATAGAGCAGCTGGACAACCAGCAGGTGATAGAGCAGCTGGACAACCAGCAGGAGAT A G D R A A G Q P A G D R A A G Q P A G D AGCAGATGGAC^GCCAGCAGGAGATAGAGCAGCTGGACAGCCAGCAGGAGATAGAGCAGATG A D G Q P A G D R A A G Q P A G D R A D CAGCCAGCAGGAGATAGAGCAGCTGGACAGCCAGCAGGAGATAGAGCAGATGGACAGCCAGC Q P A G D R A A G Q P A G D R A D G Q P A GAGATAGAGCAGCTGGACAGCCAGCAGGAGATAGAGCAGCTGGACAGCCAGCAGGAGATAGA G D R A A G Q P A G D R A A G Q P A G D R AGCTGGACAGCCAGCAGGAGATAGAGCAGCTGGACAGCCAGCAGGAAATGGTGCAGGTGGAC A G Q P A G D R A G Q P A G N G A G G GCAGCAGGAGGAAACGCAGGAGGACAGGGACAAAATAATGAAGGTGCGAATGCCCCAAATG A A G G N A G G Q G Q N N E G A N A P N GAAA G T CΓGTGAAAGAATACCTAGATAAAGTTAGAGCΓACCGTTGGCACCGAATGGACTCCAT E K S V K E Y L D K V R A T V G T E W T P CAGTGTAACCTGTGGAGTGGGTGTAAGAGTCAGAAGCAGAGTTAATGCAGCTAACAAAAAACC S V T C G V G V R V R S R V N A A N K K P i GAGGATCTTACΓTTGAATGACCΓTGAGACΓGATGTTTGTAC^^TGGATAAGTG ΈΓ D T L N D E T D V C T M D K C A G I TTAICGTTGTGAGTAATTCATTAGGGCTAGTCATATTGTTAGTCCTAG<-ATTATTC-AATTAA Γ N V V S N S L G L V I L L V L A L F N [c-terminus] IΌ The DNA fragment chosen for insertion in the yeast ho was: GCAGAACCAAAAAATCCACGTGAAAATAAGCTGAAGCAACCAGGAGACAGAGCAGATGGACAG . E P K N P R E N K L K Q P G D R A D G Q CAGCAGGAGACAGAGCAGATGGACAGCCAGCAGGTGATAGAGCAGATGGACAACCAGCAGGAG 15 P A G D R A D G Q P A G D R A D G Q P A G TAGAGCAGCTGGACAACCAGCAGGAGATAGAGCAGATGGACAGCCAGCAGGAGACAGAGCAGA R A A G Q P A G D R A D G Q P A G D R A D GGACAGCCAGCAGGAGACAGAGCAGATGGACAACCAGCAGGAGACAGAGCAGATGGACAACCA G Q P A G D R A D G Q P A G D R A D G .Q P 20 CAGGTGATAGAGCAGCTGGACAACCAGCAGGTGATAGAGCAGCTGGACAACCAGCAGGAGATA A G D R A A G Q P A G D R A A G Q P A G D AGCAGATGGACAGCCAGCAGGAGATAGAGCAGCTGGACAGCCAGCAGGAGATAGAGCAGATGG A D G Q P A G D R A A G Q P A G D R A D G CAGCCAGCAGGAGATAGAGCAGCTGGACAGCCAGCAGGAGATAGAGCAGATGGACAGCCAGCA 25, P A G D R A A G Q P A G D R A D G Q P A GAGATAGAGCAGCTGGACAGCCAGCAGGAGATAGAGCAGCTGGACAGCCAGCAGGAGATAGAG G D R A A G Q P A G D R A A G Q P A G D R AGCTGGACAGCC.-AGCAGGAGATAGAGCAGCTGGACAGCCAGCAGGAAATGGTGCAGGTGGACA A G Q P A G D R A A G Q P A G N G A G G Q 3O GCAGCAGGAGGAAACGCAGGAGGACAGGGACAAAATAATGAAGGTGCGAATGCCCCAAATG A A G G N A G G Q G Q N N E G A N A P N AAAAGTCΓGTGAAAGAATACCΓAGATAAAGTTAGAGC?ΓACCGTTGGCACCGAATGGACTCCA E S V E Y L D K V R A V G T E W T P This fragment includes the entire tandem repeat s 35 quence plus a region that is substantially parallel to Region of Dame, et al Science 225:628 (1984), precedes the repeat r gion, and codes for the amino acid sequence A E P K N P R E N L K Q P G. This sequence includes the subsequence K L K Q P wh is conserved in all malarial species that have been inv tigated to date. The foregoing DNA fragment was obtained from the ent gene by subcloning a 15kb Bglll fragment isolated as descri by Arnot et. al (U.S. Patent Application Serial No. 754,6 and Science 230:815-818. November 15, 1985) both incorpora by reference. It was then inserted in the DNA of modif yeast plasmid pAB24 as described in Example 1 below. For expression of the P. vivax CS antigen in yeast hybrid promoter comprising the strong yeast glyceraldehyde phosphate dehydrogenase and the glucose regulatable alco dehydrogenase-2 (ADH-2) promoter was used. Fusion of this p moter to heterologous genes allows the growth of yeast cultu to high density using glucose as a carbon source. Depletion glucose in the media during fermentative growth leads concomitant induction of expression of the heterolog protein. Incorporation of the plasmid into high copy numb autonomously replicating yeast plasmids, and transformation yeast cells generated strains capable of expressing high lev of CS proteins on induction. The yeast was grown in culture in YEP medium (1% yeast extract, 2% peptone) with 1% glucose as described Example 1 below. Two hundred liters of yeast material w thus obtained and stored at - 80 *C. The thus obtained yeast material contained a comp mixture of different yeast proteins as well as culture med additives. Gel electrophoresis of this material on 7.5% sod dodecyl sulfate polyacrylamide gel gave an indication of heterogeneity (see Figure 6, lane l) . The expression proced and plasmid construction are outlined in Figure 1 and descri in Example 1, below. It had been observed that all antibodies to the knowlesi CS protein recognized the immunodominant epit region of this protein even after the CS protein had b heated at 100*C for 30 minutes or subjected to compl denaturation by treatment with 6M guanidine and 1% beta mercaptoethanol (Gysin, J., et al £,_ Exp. Med. 16_0_:935, 1984 Godson, G.N., et al Nature 3_£5_:29, 1983). This observation concerned the entire P. knowlesi protein and by no means established either that the P.vivax protein would show similar behavior upon heating or that fra ments thereof, consisting essentially of the polypepti employed herein and encompassing the epitope region, wou continue to be immunochemically reactive with anti-P.vivax protein antibodies, after being subjected to heating conditio that normally result in denaturation of proteins. Anoth imponderable was that, in the present invention, the fragme of the CS protein was mixed with very large amounts of no relevant materials. Heating of the mixture might lead to t formulation of aggregates with other polypeptides and maski of the epitopes and/or coprecipitation. It was also not kno whether such fragments would continue to be immunogenic aft being subjected to the above heating treatment. Nevertheless, the present inventors used a heating st as the initial step in the purification of the yeast-engineer P.vivax CS polypeptide. (As used in this patent applicatio "polypeptide" will refer to a relatively long protein fragmen "protein* will refer to the entire protein, and "peptide" wi refer to a relatively short peptide, e.g., one containing amino acid residues or less. Regarding the use of the wo "sequence", it will be understood that polypeptides a peptides in accordance with the present invention will functionally equivalent if they have the same sequence wheth the sequence is set forth from the N to the C terminus or fr the C to the N terminus) . The mixture was then subjected to heating at 100* which resulted in massive precipitation of lysed yeast cell m terial. The supernatant was separated, dried and lyophilize The lyophilized supernatant residue showed a substanti improvement in purity over the yeast extract (see Fig. 6, la 4). A solution of the lyophilized material was then furth purified sequentially by (a) anion-exchange chromatogr using an electrolyte gradient to elute the engineered polypeptide; and (b) molecular sieve chromatography. The fractions containing CS polypeptide activity identified with a radioimmunoassay. A small amount of highly purified yeast material was radiolabelled to a h specific activity with 125I. Each fraction was assayed b classical radioimmunoassay for its capacity to inhibit binding of the labelled material to immobilized anti-P.vi monoclonal antibody, directed against the repetitive epitope the P.vivax CS protein. A major advantage of the purification process of present invention is its simplicity and its ready adaptabil to scale-up. The thus purified engineered P.vivax CS polyp tide is homogeneous by SDS-PAGE and isoelectric focusing and thus expected to be substantially free of pyrogenic, infl matory and toxic impurities that may have been associated w the lysed yeast cell material and yeast culture media. The yield of the combination of the yeast express procedure and the purification process of the present invent proved to be 13 mg of pure CS polypeptide per liter of ye culture. Given that 200 liters of this culture were produ in three days using pilot scale equipment (250 liter f enter) , it is apparent that large amounts of the engineered polypeptide can be made available in a short period of time. A 200-liter stock of yeast extract contains suffici engineered CS polypeptide to immunize about 25,000 hum against P.vivax. using 100 micrograms of polypeptide pe son. Major advantages of the CS polypeptide produced accordance with the present invention include that the gineered peptide: (a) can be produced in large scale free of impurit and of non-relevant antigens; (b) represents a large fragment of the native CS mo cule; (c) has been shown to be highly immunogenic in rode using aluminum hydroxide as an adjuvant; (d) the antibodies which are produced react with bo the "repeats" and a conserved region of the CS molecule. Antibodies to the "repeats" have been shown to neutra ize parasite injectivity very effectively. Antibodies to peptide containing this conserved region also neutraliz parasite injectivity, but the inhibitory activity could not accurately quantitated (Vergara, et al, £. Immunol. 134: 344 3448, 1985). Moreover, the fact that the sequence KLKQP, whi is part of the peptide, is present in all CS proteins, sugges that this region has an important function. The present invention is described in detail below specific examples which are intended to illustrate the inve tion without limiting its scope. EXAMPLE_ L: Restriction of the P.vivax CS gene, Ligation int a Vector, Transformation of the Host Yeast Cells and Expression In the following description all restricti endonucleases and enzymes are commercially . available fr Pharmacia Fine Chemical Co., (Piscataway, N.J.), New Engla Biolabs (Beverley, MA) , Boehringer Mannheim (Indianapolis, I or Bethesda Research Laboratories, Inc. (Gaithersburg, MD) a are used according to the manufacturer's instruction. A pUC9 vector (Pharmacia Fine Chemical Co., Piscatawa NJ) containing the P. vivax CS protein gene, was derived subcloning a 15-kb Bglll fragment inserted into the BamHI sit of EMBL3, a bacteriophage lambda vector (as disclosed in Arn et. al., U.S. Patent application serial number 754,645 inco porated by reference) . Clone pUC9Ci was digested with Bgl and XBal and gel-purified to obtain a 4.1kb fragment encodi all the repeat sequences (Arnot et. al., Science 230:815 - 81 1985, incorporated by reference.). This gel-purified fragme was then digested with Fokl and BanI and ligated to t following 5'-phosphorylated synthetic linkers synthesized phosphoramidite method using Applied Biosystems' 380A D synthesizers. I CCTTGGAACCATGG GGAACCTTGGTACCGTCT Ncol Complementary to Fokl overhang II GCACCGAATGGACTCCATAG GCTTACCTGAGGTATCCAGCT BanI Sail Linker I provides for an Ncol site, while Linker provides a Sail overhang. The linker-containing fragment was digested with N and Sail, isolated by gel electrophoresis and cloned onto Nc Sail digested pBSlOO (construction described below) . resulting plasmid is designated pAG/P.vivaxl. pBSlOO is pBR322-derived plasmid containing the ADH-2 regulated GA (glyceraldehyde-3-phosphate dehydrogenase) promoter (1200 and GAPDH terminator (900bp) (See Figure 1) and its constr tion is described below. The ADH-2 portion of the promoter was constructed cutting a plasmid containing the wild type ADH-2 gene (plas pADR2, see Beier and Young, Nature. 300:724 - 728 (1982) corporated by reference with the restriction enzyme Eco which cuts at a position +66 relative to the ATG start cod as well as in two other sites in pADR2, outside of the AD region. The resulting mixture of a vector fragment and smaller fragments was digested with Bal31 exonuclease to rem about 300bp. Synthetic Xhol linkers having the sequence C C T C G A G G G G A G C T C C were chemically synthesized and ligated onto the Bal31 trea DNA. The resulting DNA linker vector fragment (about 5kb) separated from the linkers by column chromatography Sepharose CL4B from Pharmacia) , cut with the restriction enz Xhol, religated and used to transform E_t. colj to ampicil resistance. The positions of the Xhol linker additions w determined by DNA sequencing using standard techniques w known in the art. One plasmid which contained an Xhol lin located within the 5' non-transcribed region of the ADH-2 g (position -232 from ATG) was cut with the restriction enz Xhol, treated with single-strand specific nuclease Si, a subsequently treated with the restriction enzyme EcoRI create a linear vector molecule having one blunt end at t site of the Xhol linker and an EcoRI end. The GAP portion of the promoter was constructed cutting plasmid pPGAP (as disclosed in European Patent Applic tion No. 164,556 of Chiron Corporation filed on May 3, 198 incorporated by reference and accorded the filing date of corresponding U.S. application Serial No. 609,540 filed on M 11, 1984) with the enzymes BamHI and EcoRI, followed by t isolation of the 0.4 bp DNA fragment. Plasmid pPGAP is a yea plasmid containing a yeast GAPDH promoter and terminat sequences with flanking Ncol and Sail restriction endonuclea sites. The purified fragment was partially digested with t enzyme Alul to create a blunt end near the BamHI site and us to construct plasmid pJS104. Plasmid pJS104 was constructed by the ligation of t AluI-EcoRI GAP promoter fragment to the ADH-2 fragment prese on the linear vector described above. The BamHI-Ncol ADH-GAP promoter fragment was obtain from plasmid pJS103, which is the same as pJS104 (supra) exce that the GAP fragment of the ADH-GAP promoter is about 200 in pJS103 and 400 bp in pJS104. Construction of pJS103 was t same as that for pJS104 except that the 0.4 kb BamHI-Eco fragment was completely digested with Alul (instead of partia ly digested for pJS104) and a 200 bp fragment was isolated. The entire above region containing the promoter, vivax segment and terminator, (hereinafter termed the "expre sion cassette") was excised by digestion with BamHI, purifi by gel electophoresiε and cloned into BamHI-digested pAB2 . restriction map of this plasmid is shown in Figure 2. pAB24 is a yeast expression vector (Fig. 2) which co tains the complete 2 mu sequences necessary for an autonomo replication in yeast (Broach, in: Molecular Biology of t Yeast Saccharomvces. 1= 45, Cold Spring Harbor Press, 1981) a pBR322 sequences. It also contains the yeast URA3 gene deri from plasmid YEp24 (Botstein, et al.. Gene (1979) 8_:17 inco porated by reference) and the yeast LEU2 d gene derived plasmid pCl/1 (see European Patent Application Serial 116,201 filed on August 22, 1984 in the name of Chiron Corp tion, incorporated by reference) . Insertion of the expres cassette was in the fi HI site of pBR322, thus interrupting gene for bacterial Resistance to tetracyclin. Expression of P. vivax CS proteins Saccharomyces cerevisiae strain AB110 isolated Chiron Corporation (Mat. leu2-04. or both leu2-3 and leu2-1 pep4-3. his4-580. cir # ) was transformed with pAB24/£. viva according to Hinnen, et al., Proc. Natl. Acad. Sci. USA 75:1 - 1933 (1978). Single-transformant colonies harboring G regulated vectors were grown in 2ml of leu" (leucine-deplet selective media to late log or stationary phase. Only ye harboring the plasmid can grow in this medium. Cultures w subsequently diluted 1:20 (v/v) in YEP (1% w/v yeast extra 2% w/v peptone) with 1% glucose, and grown to saturation (ab 36h) in this medium. Cells were lysed in the presence sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) and lysates were clarified by centrifugation. Cleared lysates w subjected to polyacrylamide gel electrophoresis (Laemmli, U. Nature. 277:680, 1970). Following staining with Coomas blue, a heavy band of about 38kD was observed in extracts f transfor ants containing the P. vivax plasmid (Figure 3) . T band was detected in those cells transformed with the expr sion vector, while being absent from extracts of cells harb ing control (pCl/1) plasmids. The fusion protein accounts over 10% of the total cell protein. The reason for abnor cell migration (38kD versus 23kD, predicted from DNA constr tion) may be attributable to anomalous SDS binding as previo ly reported for P. Knowlesi CS proteins (Ozaki, et al., C 3_4_!185, 1983), probably due to the low proportion of hyd phobic residues. To confirm the identity of the 38kD band, prote synthesized by yeast were also submitted to Western analys Cleared yeast lysates prepared as described above were elect phoresed on polyacrylamide gels (Laemmli, supra) and prote were subsequently electroblotted onto nitrocellulose filt (Towbin, et. al., Proc. Natl. Acad. Sci. USA 76:3450, 1979 The filter was preincubated for lh with 1% (BSA) in PBS subsequently treated with a monoclonal antibody to P. vivax protein for 12h at 4'C. The filters were washed with BSA/PBS and a second goat anti-mouse antibody conjugated w horseradish peroxidase (BioRad Laboratories, Richmond, Calif nia) added. Finally, the filters were incubated with hors radish peroxidase color development reagent (Bio-Rad, Richmo CA) and washed. The western analysis showed that the fus protein reacted with the monoclonal antibodies. (Fig. 4) EXAMPLE 2: Purification of the circumsporozoite polypept of the Plasmodium vivax The yeast cultures expressing a part of the circ sporozoite polypeptide were prepared as in Example 1. The pressed polypeptide consisted of 234 amino acids including of the repeat domain and a conserved region, namely Region I Dame, J.B., et al, Science 225:628 (1984). The N-termi amino acid of the expressed polypeptide is alanine iri nucleotide positions No. 385-7 (GCA) and the C-terminal am acid is proline at nucleotide positions No. 1084-1086 (CC Arnot, et al, Science 230:815-818. 1985. The first step purification of the peptide fragment expressed by yeast c sisted of subjecting the extracts to temperatures of 100* The purification was performed as follows: Extracts from pelleted yeast from 20 liters of ye culture were prepared in a bead beater, diluting the yeast an equal volume of 0.1 M sodium phosphate buffer, pH 7.3, 0. Triton-X, 1 mM EDTA, 1 mM PMSF (phenylmethylsulfonylfluori and 1 microgram per ml of pepstatin. The extract (370 ml) added to 200 ml of boiling water containing 1 mM of PMSF an microgram/ml of leupeptin. The mixture was brought to temperature of 100 * C and kept for ten minutes with const stirring at this temperature. The mixture was cooled to and centrifuged at 18,000 rotations per minute in a Beck Ultracentrifuge Rotor TI-19, (Beckman Instruments, Palo Al CA) for fifteen minutes. The supernatant was removed and t lyophilized. The dry material was dissolved in 120 ml of wat centrifuged to remove a small residual amount of insolu material, dialyzed extensively against distilled water for hours and lyophilized again. The powder was dissolved in 3 sodium potassium phosphate buffer, pH 7.5, and the conductiv adjusted with water to 0.58 mS. The solution was centrifu to remove insoluble materials and subjected to anion excha chromatography in a DEAE-Sephacel (Pharmacia Fine Chem C Piscataway, N.J.) column (5cm x 24cm) equilibrated in the s buffer. The flow rate was adjusted to 100ml per hour and 21 per tube were collected. The column was then washed with ml of the same buffer, i.e., with about one column volume. elution continued with a buffer formed in a linear gradient which 1,500 ml of the initial buffer were gradually mixed w 1,500 ml of the same buffer also containing 0.75 M NaCl. presence of the circumsporozoite polypeptide in the vari fractions eluting from the column was detected using a compe tive radioimmunoassay described below. The positive fractions eluted between fraction Nos. 90 in a symmetrical peak with conductivities between 2 and mS (Fig. 5) . The full fractions 65-90 were lyophiliz redissolved in 60 ml of 0.3 M NaCl and dialyzed against 0. NaCl at room temperature for several hours. The dialyzate centrifuged to remove a small amount of insoluble material one third, i.e. 20 ml, was subjected to molecular si chromatography on Sephadex G-200 (Pharmacia) equilibrated w 0.3 M sodium chloride. The Sephadex was superfine and column was 5 cm in diameter and 100 cm long. Samples of 21 per tube were collected from the column. The CS polypept eluted in a sharp symmetrical peak between tubes 51- However, materials with higher and lower molecular weight smaller amounts were distributed in tubes preceding following this peak. The contents of tubes 51-57 were pool dialyzed extensively against distilled water, and lyophiliz A total amount of 89 mg of pure circumsporozoite polypepti were recovered. On this basis, we calculated that the yi from 20 liters of yeast is 89 x 3, i.e., 267 mg of pure prote or about 13 mg per liter of yeast culture. The purity of t recovered circumsporozoite polypeptide was assessed by sodi dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAG and by isoelectric focusing under denaturing conditions. concentrations of 2 eg per ml, a single band with an is electric point of 4.3 was detected by isoelectric focusi (Fig. 7) . By SDS-PAGE, a doublet was detected with a molecul weight between 43,000 and 45,000 (Fig. 6). The extincti coefficient at 280 nanometers of solutions of the CS polype tide containing 1 mg of protein per ml was 0.2. A sample the polypeptide was subjected to partial N-terminal sequen analysis and the major sequence was alanine-glutamic aci proline-lysine-asparagine-proline, as expected. Another samp was radiolabelled with 125I and immunoprecipitated with mon clonal antibodies specific to the CS protein of Plasmodi vivax. Between 75 and 85% of the counts were specifical recognized by the antibody. A solid state competitive radioimmunoassay was usέd detect the engineered CS polypeptide during the purificati procedure. The standard curve relating the dose of antig with the signal obtained in the radioimmunoassay was prepar as follows. The purified, engineered CS polypeptide w radiolabelled with 125I to a specific activity of about 2 x 1 counts per minute per microgram protein using the well-kno iodogen method. Mixtures containing a constant amount radiolabelled engineered CS polypeptide and variable amounts purified cold (i.e. unlabeled) engineered CS polypeptide (in total volume of 100 microliters) were prepared. Thir microliters of the mixtures were then delivered to the bott of wells of microtiter plates pre-coated with monoclon antibodies to the circumsporozoite protein of Plasmodium viv (2F2) . This monoclonal antibody reacts with the repetiti epitope of the circumsporozoite protein. (Nardin E.H. et a J_. EXP. Med.. 156:20. 1982). The inhibitory effect of t cold peptide on the binding of the labeled peptide to t bottom of the wells was proportional to the concentration cold peptide. This assay detected concentrations of c peptide as low as five nanograms per ml (Fig. 8) . The assay the column fractions and of the extracts was performed exac as described above and the degree of inhibition obtained referred to the standard curve (Fig. 8) to calculate concentration of the CS protein peptide. All dilutions washing in this assay were performed in a phosphate buf saline (PBS) containing 1.0% bovine serum albumin (BSA) 0.1% sodium azide. On the basis of the results of these assays, we cal lated that the yeast cultures contain about 60 mg of c cumsporozoite protein per liter and that the recovery of purified material was about 20% of the total. There were losses in the initial step of purification, i.e., following boiling of the extracts this step removed more than 90% of non-relevant protein bands observed in the SDS-PAGE of original extracts. FYΆMPT.TC I; Immunization of mice with the yeast-engineered P.vivax polypeptide The purified engineered CS polypeptide was then used an antigen to immunize mice. Ten outbred female Swiss-Webst 8-12 week old mice were injected with 50 miσrograms of purified peptide adsorbed on aluminum hydroxide. Three and weeks afterwards, the mice were boosted with the same dose antigen again using aluminum hydroxide as adjuvant. Ten d later, the mice were bled and the sera were subjected to an ysis by an immunoradiometric assay to detect antibodies to CS polypeptide. In this assay, the wells of microtiter pla are coated with purified engineered CS polypeptide (10 mic grams/ml in PBS) and then incubated the wells with 30 mic liters of various dilutions of the mouse serum in PBS-BSA. wells were then washed and re-incubated with 50,000 cpm radiolabelled affinity-purified goat anti-mouse immunoglobu (10 7 cpm/microgram of protein) diluted in PBS-BSA. The we were then washed again and counted. All sera reacted with the engineered CS polypeptide dilutions of 1:10,000 or greater. The results of a titrat of the pooled sera are shown in Fig. 9. That these se contained large amounts of antibodies to the "repeats* of t P. vivax CS protein was shown in two experiments describ below. 1) A synthetic 18-amino acid peptide (18-mer) co prising the sequence (Asp-Gly-Gln-Pro-Ala-Gly-Asp-Arg-Ala) 2 a representing two tandem repeats of the P. vivax CS Protein w synthesized as described in (a) copending U.S. patent applic tion Serial No. 754,645 filed on July 9, 1985 and incorporat by reference in the present application; and (b) Arnot, et a Science. 230:815, 1985. This peptide was used to coat wells microtiter plates and the antibody contents of the se detected by the immunoradiometric assay described above. A sera reacted with titers at 1:4,000 or above. The results titrations of pooled sera obtained after the second antig booster injection are shown in Fig. 10. 2) The synthetic 18-amino acid peptide was also us to inhibit the binding of antibodies, present in the pool antiserum, to the wells of plastic plates coated with t engineered CS polypeptide. In these experiments, we fir incubated samples of a 1:3,000 dilution of the pooled ser from the mice (obtained after the 2nd booster rejection) wi increasing concentrations of the 18-amino acid pepti described above. As a control, samples of serum were incubat with the same concentration of another 18-mer peptide with different and unrelated sequence of amino acids. Following incubation of one hour at room temperature, 30 microliters the mixtures were added to wells of plates precoated with t engineered CS polypeptide. The wells were then washe incubated with radiolabelled goat anti-mouse immunoglobulin, described above, washed again and counted. The results sho in Fig. 11 demonstrated that the "repeat" peptide (but not control peptide) inhibited about 50% of the reaction betw the antibodies and the CS protein. All dilutions and washi in these experiments were performed with PBS-BSA. The same sera were also analyzed for the possible p sence of antibodies to Region 1 of Dame, et al., supra. this purpose, we used as an immobilized antigen a s synthetic peptide NH 2 -Cys-Tyr-A^n-5Jji-L^s-IlS-illu-Ar5- n- Lys-Leu-Lys-Gln-Pro-COOH. This peptide includes a sequence five amino acids which are common to all CS proteins from malaria parasites examined to date, Lys-Leu-Lys-Gln-Pro ( et al., Science. 225:593. 1984, and Enea, V., personal c munication) . The first two amino acids (Cys and Tyr) extraneous to the CS protein and were added for purposes coupling the peptide to a carrier protein and radiolabell with 125I. This peptide was used to coat wells of mi.croti plates and the antibody contents in the sera were detected described above. After the second booster, all sera recogni this small peptide at dilutions of 1:2,000 or greater. results of titrations of a pool of these sera is shown in F 12. The sera were also tested by indirect immunofluo scence using the sporozoite of P. vivax as the antigen. sera were positive at dilutions of 1:1,000 or greater. Finally, another experiment demonstrated that re tively low levels of antibodies to the engineered CS prot neutralize the infectivity of P. vivax sporozoites. The as was performed as described in J_. Immunol. 132:909, 1 (incorporated by reference) using the human hepatoma cell l Hep 62 as the target of parasite invasion. The results, forth in Table I, below, show that a significant degree inhibition was obtained at serum dilutions of 1:5,000. percentages of inhibitions were calculated as 100-[(m experimental values/mean of controls)xlOO] . Controls consis of sporozoites incubated with equivalent concentrations normal (pre-immune) mouse serum. TABLE I INHIBITION OF P. VIVAX SPOROZOITES INTO HEPA- TOCYTES IN VITRO BY ANTIBODIES TO THE YEAST- ENGINEERED CS PRO EIN (Serum Dilution^ " %Inhibition 50 91 250 91 1,000 77 5,000 42

Claims: WHAT I CLAIM IS : 1 1. A method for purifying a polypeptide consisting 2 essentially of an amino acid sequence consisting essentially of 3 at least two tandemly repeating sequences idential to those of 4 the immunodominant epitope region of the circumsporozoite 5 protein of P.viva , from an impure preparation of said polypep- 6 tide said impure preparation also containing one or more other 7, protein species as impurities, said method comprising the steps 8 of: g. (a) subjecting said preparation to a temperature of IS. 100 C for a period of time sufficient to cause TT: denaturation and precipitation of the bulk of said r protein impurities, while leaving said polypeptide 13 in solution; 14- (b) removing and discarding said precipitate and 15 retaining the supernatant; 16 (c) separating the solid constituents of said s per- 17 natant; 18 r ' (d) redissolving said solid constituents in an aqueous 13 ionic buffer solution; 20 (e) subjecting said solution to ion-exchange 21 chromatography on a first chromatography column, 22. thereby causing said polypeptide to be retained on said 23 column, while a portion of the remaining impurities of 24 said polypeptide are excluded in the effluent, and 25- eluting said polypeptide using an ionic gradient; 26 (f) decreasing the ionic strength of said eluant 27 containing said polypeptide; 28- (g) subjecting " said eluant to a molecular-sieve 29 chromatography on a second chromatography column, and 3D eluting said polypeptide from said coϊum. 1 2. The method of claim 1 wherein said polypeptide is 2 a yeast-engineered polypeptide having an amino acid sequence 3, consisting essentially of the immunodominant epitope region of 4 the circumsporozoite protein of Plasmodium vivax flanked by 5 amino acid sequences contiguous to said region. 3. The method of claim 2 further comprising separa ing said supernatant from said precipitate by centrifugati followed by at least one lyophilization of said supernatant a subsequent redissolution of the lyophilized material, prior said first chromatography step. 4. The method of claim 3 further comprising eluti said polypeptide from said first column using a linear Na gradient of increasing ionic strength. 5. The method of claim 3 further comprising decrea ing the ionic strength of said eluant by dialysing said elua against a buffer of lesser ionic strength than said eluant. 6. A synthetic polypeptide comprising at least o epitope of the circumsporozoite protein of P.vivax other th the immunodominant epitope which consists of a repeati sequence of amino acids, said polypeptide being expressed by recombinant organism and having an amino acid sequence contai ing: (a) a repetitive amino acid sequence of said ci cumsporozoite protein which represents a repeati immunodominant epitope region of said protein; and (b) a portion of the amino acid sequence of t circumsporozoite protein of Plasmodium vivax whi immediately precedes the repeating epitope region said protein proximal to the N-terminal of said prote and which is conserved in the CS proteins of differe malarial species. 7. The polypeptide of claim 6 having the property conferring protective immunity against Plasmodium viv sporozoites to a mammalian host susceptible to infection wi P.vivax sporozoites. 8. The polypeptide of claim 6 wherein said sequen in (b) precedes the repeating sequence and contains the amin acid sequence KLKQP. 9. The polypeptide of claim 6 wherein said repetitiv sequence is (DRAXGQPAG) r wherein X is D or A and r is a integer between 2 and 19 inclusive. 10. The polypeptide of claim 9 wherein said repetitiv sequence is of the same length as the entire immunodominan epitope region of the native P. ivax circumsporozoite protein. 11. The polypeptide of claim 6 also comprising segment of the amino acid sequence of the circumsporozoit protein of Plasmodium vivax following the repeating epitop region of said protein towards the C-terminal end of sai protein which segment is conserved in the CS proteins of dif ferent malarial species and located at the C-terminal of sai polypeptide. 12. The polypeptide of claim 6 also comprising th entire segment of the amino acid sequence of the circum sporozoite protein following -the repetitive immunodominan epitope region and extending to the C-terminal of said protein 13. A synthetic peptide comprising the amino acid KLKQP in sequence, said amino acid sequence being conserved i the CS protein of Plasmodium knowlesi, Plasmodium falciparu and Plasmodium vivax. 14. A synthetic polypeptide having an amino aci sequence consisting essentially of (a) an immunogenic repetitive sequence co responding to all or part of the repetitive i munodominant epitope region of P.vivax circumsporozoi protein; and (b) located in at least one of the N- and terminal of said repetitive sequence, a non-repetiti 25 • amino acid sequence which in the native ci cumsporozoite protein (1) immediately precedes follows the repetitive immunodominant epitope region said protein, and (2) is conserved among differe malarial species. 15. The polypeptide of claim 14 wherein said sequen in (b) is located at the N-terminal of said polypeptide and the non-repetitive conserved sequence immediately preceding t repetitive epitope region of the native circumsporozoi protein and located proximal to the N-terminal of said protei 16. A synthetic polypeptide having an amino ac sequence comprising: (a) an immunogenic repetitive sequence co responding to the repetitive immunodominant ' epito region of P.vivax circumsporozoite protein; and (b) at the N-terminal of said repetitive sequen a non-repetitive amino acid sequence which in t native circumsporozoite P.vivax protein (1) immediate precedes and is located at the 5'-end of the repetiti immunodominant epitope region of said protein, and ( is conserved among the different malarial species. 17. The polypeptide of claim 6 wherein said sequen (b) is located at the N-terminal of said polypeptide.

Inventors: NUSSENZWEIG VICTOR, NUSSENZWEIG, VICTOR
Applicants: UNIV NEW YORK, NEW YORK UNIVERSITY
Application year: 1988
Main classification: C07K3
IPC codes: A61K39, A61P33, C07K7, C07K14, C07K19, C12N15, C12P21, C12R1