WO1993024000A1 - Procedes d'obtention et de purification de proteines de surface pneumococciques sans cellules a partir de s. pneumoniae et leur utilisation - Google Patents

Procedes d'obtention et de purification de proteines de surface pneumococciques sans cellules a partir de s. pneumoniae et leur utilisation Download PDF

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Publication number
WO1993024000A1
WO1993024000A1 PCT/US1993/005191 US9305191W WO9324000A1 WO 1993024000 A1 WO1993024000 A1 WO 1993024000A1 US 9305191 W US9305191 W US 9305191W WO 9324000 A1 WO9324000 A1 WO 9324000A1
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Prior art keywords
choline
protein
pspa
streptococcus pneumoniae
solution
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Application number
PCT/US1993/005191
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English (en)
Inventor
Janet Yother
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Janet Yother
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Publication date
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Publication of WO1993024000A1 publication Critical patent/WO1993024000A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention concerns methods for causing
  • Streptococcus pneumoniae strains to release substantial quantities of their surface proteins, in particular pneumo ⁇ coccal surface protein A (PspA) , into a medium and methods for purification and recovery of the surface protein so produced.
  • the surface protein may be used with or without
  • Streptococcus pneumoniae is an important cause of otitis media, meningitis, bacteremia and pneumonia. De-
  • mice described immunization of mice with a recombinant full length fragment of PspA which elicited protection against strains of pneumococcal capsular types 6A and 3.
  • Crain et al. Infect. Immun. 58.:3293-3299 (1990) describe a rabbit antiseru that detects PspA in 100% of clinical and laboratory isolates of S. pneumoniae strains. Fifty-seven isolates reacted with seven monoclonal antibodies to PspA exhibited thirty-one different patterns of reactivity.
  • PspA protein type is independent of capsular type. It appears that genetic mutation or exchange in the environment has allowed development of strains which are highly diverse with respect to capsule, PspA, and possibly other molecules as well. PspAs from various strains vary in their molecular weights, ranging from 67 to 99 kD. These differences are inherited stably and do not result from protein degradation. While PspA varies in structure between different pneumoccal strains, all PspAs exhibit 5 numerous cross-reactions, which suggests that sufficient common epitopes may exist to allow a single PspA or a small number to elicit protection against a large number of S. pneumoniae strains.
  • McDaniel et al., McDaniel IV described a recombinant full- length PspA and its expression in E. coli.
  • the supernatants of lysates from the modified E. coli were used for immunizations without further purification.
  • the PspA expressed in E. coli were localized to the periplasmic space from which they could be released by standard osmotic shock procedures.
  • the isolated truncated segments contain immunoprotective epitopes of the protein, but lack the region which anchors the protein to the cell
  • S. pneumoniae appears to be unique among gram positive bacteria in having surface proteins, in particular PspA,
  • each R is individually selected from the group com ⁇ prising methyl, ethyl, hydroxyethyl and esterified hydroxy- ethyl radicals; and R' is methyl or hydrogen.
  • DEAE diethylaminoethanol
  • cultures of S. pneumoniae can be caused to express substantially cell-free surface proteins, in particular PspA, into a medium from intact 5 cells either during growth of a culture or thereafter.
  • the supernatant medium may be used directly as an immunogen or may be used for preparation of more purified immunogens, undesired materials may be removed before use, or the PspA in the supernatant may be separated and purified as needed.
  • PspA pneumococcal surface protein A
  • choline-bindable proteins may be freed from the walls of intact cells of S. pneumoniae by exposing the cells during growth or thereafter to a medium comprising from about 0.5% to about 3% or more of choline or a choline ana-
  • the surface protein-containing medium is then sepa ⁇ rated from the cells by any convenient means such as decantation, etc. and may then be freed of cells and cellular debris by means such as filtration or centrifugation in the usual manner.
  • the resulting solution is then sepa ⁇ rated from the cells by any convenient means such as decantation, etc. and may then be freed of cells and cellular debris by means such as filtration or centrifugation in the usual manner.
  • choline-bound proteins i. e. proteins which had been bound to choline residues in the cells
  • they may then be used as such as a vaccine or to induce the formation of antibodies in animals to provide passive protection against pneumococcal infection, or they may be further treated as
  • a particular method for practice of the invention is to cause S. pneumoniae to express surface proteins which are ordinarily bound by interaction with choline residues of the S. pneumoniae cell membrane or wall by growing the
  • % choline cell-associated PspA cell-free PSPA about 0.05% about 98% about 2% 0.1% about 50% about 50%
  • CDM chemically defined medium
  • Other culture conditions may be chosen from those well known to the art. When grown under these conditions S. pneumoniae releases choline-bound surface proteins into the supernatant. The time to reach saturation is dependent upon the strain of S. pneumoniae and the culture conditions selected. At 37 ⁇ C the doubling 5 time in CDM with 2% choline ranges from about 30 minutes to 1 hour.
  • the supernatant may be separated from the bulk of the cells by any convenient method such as
  • - ⁇ decantation, etc. may then be freed of cells and cellular debris by means such as filtration or centrifu- gation in the usual manner to yield a substantially cell- free PspA.
  • the growth may also be carried out by continuous or batch-wise addition of new culture medium and
  • a suitable buffer is a phosphate buffered saline with a pH of about 7.2.
  • purification may be accomplished by any of the methods known to the art, more particularly those in the references cited above.
  • a preferred method for purification of solutions of surface proteins such as PspA which bind to choline is to adsorb the proteins on an ion exchange column modified with a tertiary amine such as diethylaminoethanol (DEAE) .
  • This method is useful regardless of the source of the choline-
  • bindable surface protein Other tertiary amine choline analogs included in the previous listing derived from Sanz et al., OP. cit.. bound to a suitable support may also be used. If the choline-bindable surface protein solution contains choline, the choline should be removed by a method
  • a preferred method is the use of an equilibrated DEAE-cellulose column such as that described by Sanz et al. , OP. cit.
  • a suitable method for equilibration is the use of a solution of about 50mM Tris and about 0.15M sodium chloride, having a pH of about 7.4. 5
  • the presence of the anchor portion of a choline-bindable surface protein such as PspA causes it to be strongly adsorbed on the amine-modified column, which allows other materials in the PspA solution to be washed out of the column readily with a saline buffer such as that used to
  • choline-bindable surface protein such as PspA may then be easily and substantially quantitatively washed from the column using an aqueous wash solution containing from about 0.5% to about 4%, preferably from about 1.5% to about 2.5%, choline.
  • substantially completely elute the protein may be as little as 1/lOOth the original culture volume or less, but ultimately will depend on the strain used to grow the protein and the concentration of choline used in the wash solution.
  • the minimum amount is not critical, and can
  • the resulting protein solution may then be used as such, or if desired or necessary, it may be freed of choline by means such as dialysis against a saline buffer, as described above.
  • a culture of S. pneumoniae is grown in a medium containing only a small amount of choline, preferably less than about 0.05%.
  • the choice of medium is not critical. Suitable media include CDM with added choline or Todd-Hewitt plus yeast extract
  • the cells are harvested by any suitable means known to the art such as centrifugation at 10,000 RPM for 10 to 20 minutes and decantation of the supernate.
  • the harvested cells may be washed first with small amounts of water such as twice with 1/lOth the amount of the original culture volume, and then are washed with from about 1/20th to about one culture
  • the extract may be used without further purification or may be further purified as set out above, keeping in mind that the choline or choline analog should be removed from the solution before utilizing ion-exchange to purify the PspA.
  • a culture of S. pneumoniae is grown in a CDM in the substantially complete absence of choline or choline analogs in the presence of from about 0.005 to about 0.1%, preferably from about 0.02% to about 0.04% of ethanolamine.
  • the culture utilizes
  • the supernatant may be harvested as set out above to yield a substantially cell-
  • 35 supernatant may also be purified for further use by adsorbing the choline-bindable proteins on an amine- odified ion exchange column, eluting them with a choline wash and purifying the choline wash as set out above.
  • Application of ethanolamine supernatants to such columns does not require pretreatment such as dialysis.
  • choline-bindable protein(s) at all 5 stages of the preparation of solutions of surface-bound proteins is readily determined by methods well-known to the art such as the Western blot or silver staining of protein gels.
  • the Western blot is particularly useful for detecting PspA.
  • Silver stain of protein gels may also be """ ⁇ used to determine PspA, but the PspA of some strains such as Rxl give only faint yellowish stains.
  • the amount of PspA in supernatants of cultures grown in high choline appears to be dependent on the strain of S. pneumoniae used, but in most cases appears to be about 2 to 15 5% of the total protein. In the supernatant from one etha ⁇ nolamine culture which was studied the PspA constituted about 25% of the total protein. When a PspA solution was prepared by washing a low-choline culture of each of two strains, PspA was about 50% of the total protein. 20 The presence of PspA was determined by using silver staining and antibodies specific to PspA.
  • Whole PspA prepared according to the invention is immunogenic when injected as the PspA-containing isolate which may have undergone further purification or may be administered as the substantially cell-free supernatant. In mice, two injections of PspA are required to elicit a significant antibody response.
  • the present invention makes available PspA from strains which have not been tested previously because no truncated or full PspAs were available. Such full PspAs should elicit more protection than the two truncated PspAs and one full-length clone which were
  • lyophilized PspA-containing materials present an advantage in that they are more easily stored and shipped in closed vials. Lyophilized product may then be prepared for administration by adding a suitable carrier.
  • the immunogenically effective amount of a cell-free protein of the invention may be determined by routine experimentation. It may be administered by any of the usual means by which vaccinations are accomplished, as for example, by intradermal or subcutaneous injection, or by
  • substantially cell-free preparations of the invention may also be used to induce the production of antibodies in a suitable host without substantial modification.
  • Lyophilized protein may be used with any suitable carrier
  • This Example illustrates the method used to determine the amount of choline-bindable protein (as PspA) which was expressed by the Rxl and A66 strains of S. pneumoniae.
  • 15 Approximately 10 5 S. pneumoniae cells were inoculated into 3ml of CDM (Hazelton Research Products, Denver, PA) to which 2% choline had been added. The culture was grown standing at 37 ⁇ C overnight (about 15 hours). One ml of res spended culture was centrifuged (about 15,000g, 2 20 minutes for Rxl, an unencapsulated strain, and 5 minutes for A66, an encapsulated strain) .
  • the supernatant fluids were saved and the cells were resuspended in 1 ml of a sodium deoxycholate buffer made up to volume with sodium chloride-citrate (SSC) .
  • SSC sodium chloride-citrate
  • the cells and supernatants were 25 stored at -20"C.
  • a 20 microliter aliquot of supernatant or cell suspension was mixed with 4 microliters 5XSDS-PAGE loading buffer and boiled for 5 to 10 minutes.
  • the sample was loaded and electrophoresed through 2 30 identical 12% SDS-PAGE gels.
  • One gel was developed with silver stain, and the other was transferred to nitrocellulose and developed in a Western blot with an antibody specific for PspA.
  • silver stain does not 5 show PspA from Rxl as well as PspAs from other strains.
  • This Example is illustrative of growth in a choline-free medium.
  • Example 3 This Example illustrates growth in a low level of choline.
  • Example 1 The procedures of Example 1 were followed using the Rxl strain, except that the choline concentration was 0.0005%, and growth was not carried beyond mid-log phase to avoid the onset of lysis.
  • the cells were harvested by centrifugation, washed twice with a culture volume of water, and then once with a culture volume containing 2% choline to cause release of cellular PspA and any other choline-bound surface proteins.
  • the choline wash was filtered as above to remove any cell carry-over. Analysis of the choline wash showed that PspA represented about 50% of the total protein.
  • the Rxl strain was grown overnight using the conditions of Example 2, but using 10 ml medium.
  • the supernatant fluid was withdrawn and applied to an ion- exchange column prepared by loading a P1000 pipet tip with 200 microliters of DEAE-Affi-Blue Gel (Biorad) .
  • the inside diameter at the top of the packing was 0.5 cm, and at the bottom 0.25 cm.
  • the length was 1.7 cm.
  • the column was equilibrated with 50mM Tris, pH 7.4, plus 0.15M sodium chloride (low salt buffer) .
  • Example 5 This Example illustrates dialysis to remove choline as when it is necessary to remove choline before applying super ⁇ natant to a column according to Example 4, or if removal of choline after elution from a column is desired.
  • the sample is placed in a dialysis bag with molecular weight cut-off of more than 50,000. (If lower molecular weight molecules are to be retained, the cut-off may be lowered.)
  • Dialysis is carried out by a standard method, for example, at 4 ⁇ C in phosphate buffered saline, pH 7.2.
  • the volume of buffer to sample is about 100:1, and the buffer is changed two or three times.
  • This Example illustrates the immunogenic activity of the substantially cell-free PspA supernatant preparations according to the invention.
  • CBA/N mice were given two interperitoneal injections, two weeks apart (preliminary experimentation indicated that two injections of about 0.1 ml were required to generate an immunogenic response) , of
  • PspA of the invention with an adjuvant.
  • This Example illustrates the retention of immunogenic activity by isolated and lyophilized PspA of the invention.
  • the R36A strain of S ⁇ pneumoniae was cultured to produce substantially cell-free PspA as in Example 2 , but
  • mice 35 the concentrations shown in Table 3 and 0.1 ml was injected interperitoneally into XID mice at day 0 and day 14. The mice were then challenged with an intravenous injection of 100 times the LD 50 of WU2 strain pneumococci at day 21. none 18, 18, 25, 26, 29, 29, 68
  • Example 9 demonstrates that the non-PspA substance expressed by WG44.1 which elicited a lesser degree of protection than PspA in Example 9 appears to be protein or protein-like.
  • a substantially cell-free supernatant of a culture of strain WG44.1 (a modification of Rxl which lacks PspA), prepared according to the method of Example 1, but using 1.2% choline chloride was concentrated by ultrafiltration followed by 60% (NH 4 ) 2 S0 4 precipitation.
  • CBA/N (XID) mice were given two interperitoneal injections, two weeks apart, of 0.2 ml 10-fold concentrations of the supernatant in complete Freund's adjuvant, and then were challenged 7 days after the second injection with 100 times the LD 50 of strain WU2 pneumococci. A second group of mice was treated in the same fashion with the same supernatant preparation in adjuvant to which pronase had been added. The results are shown in Table 5.

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Abstract

L'on peut procéder à des cultures de S. pneumoniae pour exprimer des protéines de surface pratiquement sans cellules, notamment la PspA, dans un milieu issu de cellules intactes, en exposant la culture à un milieu comprenant de 0,5 % environ à moins de 3 % environ de choline, soit pendant le développement de la culture, soit après, ou en développant une culture contenant environ 0,005 à environ 0,1 % d'éthanolamine en l'absence de choline. Le milieu surnageant peut être utilisé directement pour la préparation d'immunogènes, des matériaux indésirables peuvent être éliminés après usage, ou la protéine du surnageant peut être séparée et purifiée si nécessaire.
PCT/US1993/005191 1992-05-29 1993-05-28 Procedes d'obtention et de purification de proteines de surface pneumococciques sans cellules a partir de s. pneumoniae et leur utilisation WO1993024000A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0695803A3 (fr) * 1994-05-20 1997-01-02 Uab Research Foundation Sites épitopiques de la proteine A de surface de pneumocoque
WO1998021337A2 (fr) * 1996-11-12 1998-05-22 Regents Of The University Of Minnesota Proteine de liaison c3 de streptococcus pneumoniae
AU732520B2 (en) * 1996-05-01 2001-04-26 Rockefeller University, The Choline binding proteins for anti-pneumococcal vaccines
US6245335B1 (en) 1996-05-01 2001-06-12 The Rockefeller University Choline binding proteins for anti-pneumococcal vaccines
US6592876B1 (en) * 1993-04-20 2003-07-15 Uab Research Foundation Pneumococcal genes, portions thereof, expression products therefrom, and uses of such genes, portions and products
US7078042B2 (en) 1995-09-15 2006-07-18 Uab Research Foundation Pneumococcal surface protein C (PspC), epitopic regions and strain selection thereof, and uses therefor
CN111893084A (zh) * 2020-08-21 2020-11-06 上海荣盛生物药业有限公司 肺炎链球菌高密度发酵培养基及其方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
EUR. J. BIOCHEM., Vol. 146, issued 1985, BRIESE et al., "Interaction of the Pneumococcal Amidase with Lipoteichoic Acid and Choline", pages 417-427. *
J. BIOL. CHEM., Vol. 153, issued 1944, BADGER, "The Structural Specificity of Choline for the Growth of Type III Pneumococcus", pages 183-191. *
JOURNAL BIOLOG. CHEM., Vol. 264, No. 2, issued 15 January 1989, DIAZ et al., "Subcellular Localization of the Major Pneumococcal Autolysin: A Peculiar Mechanism of Secretion in Escherichia Coli", pages 1238-1244. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6592876B1 (en) * 1993-04-20 2003-07-15 Uab Research Foundation Pneumococcal genes, portions thereof, expression products therefrom, and uses of such genes, portions and products
EP0695803A3 (fr) * 1994-05-20 1997-01-02 Uab Research Foundation Sites épitopiques de la proteine A de surface de pneumocoque
AU693175B2 (en) * 1994-05-20 1998-06-25 Uab Research Foundation Epitopic regions of pneumococcal surface protein A
US7078042B2 (en) 1995-09-15 2006-07-18 Uab Research Foundation Pneumococcal surface protein C (PspC), epitopic regions and strain selection thereof, and uses therefor
AU732520B2 (en) * 1996-05-01 2001-04-26 Rockefeller University, The Choline binding proteins for anti-pneumococcal vaccines
US6245335B1 (en) 1996-05-01 2001-06-12 The Rockefeller University Choline binding proteins for anti-pneumococcal vaccines
US6784164B2 (en) 1996-05-01 2004-08-31 The Rockefeller University Choline binding proteins for anti-pneumococcal vaccines
US7425327B2 (en) 1996-05-01 2008-09-16 The Rockefeller University Choline binding proteins for anti-pneumococcal vaccines
WO1998021337A2 (fr) * 1996-11-12 1998-05-22 Regents Of The University Of Minnesota Proteine de liaison c3 de streptococcus pneumoniae
WO1998021337A3 (fr) * 1996-11-12 1998-07-23 Margaret K Hostetter Proteine de liaison c3 de streptococcus pneumoniae
US6291654B1 (en) 1996-11-12 2001-09-18 Regents Of The University Of Minnesota Method for isolating a C3 binding protein of streptococcus pneumoniae
CN111893084A (zh) * 2020-08-21 2020-11-06 上海荣盛生物药业有限公司 肺炎链球菌高密度发酵培养基及其方法

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