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United States Patent (19) 11 Patent Number: 5,997,882 Briles Et Al

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United States Patent (19) 11 Patent Number: 5,997,882 Briles Et Al USOO5997882A United States Patent (19) 11 Patent Number: 5,997,882 Briles et al. (45) Date of Patent: *Dec. 7, 1999 54) EPITOPIC REGIONS OF PNEUMOCOCCAL Crain, M.J., Waltman, W.D. II, Turner, J.S., Yother, J., SURFACE PROTEIN A Talkington, D.F., McDaniel, L.S., Gary, B.M., and Briles, D.E., Infection and Immunity, Oct., 1990, pp. 3293–3299. 75 Inventors: David E. Briles; Janet L. Yother; Talkington, D.F., Crimmins, D.L., Voellinger, D.C., Yother, Larry S. McDaniel, all of Birmingham, J., and Briles, D.E., Infection and Immunity, Apr. 1991, pp. Ala. 1285-1289. McDaniel, L.S., Scott, G., Widenhofer, K., Carroll, J.M., and 73 Assignee: UAB Research Foundation, Briles, D.E., Microbial Pathogenesis 1986, 1:519–531. Birmingham, Ala. McDaniel, L.S., Sheffield, J.S., Swiatlo, E., Yother, J., Crain, M.J., Briles, D.E., Microbial Pathogenesis 1992, * Notice: This patent is Subject to a terminal dis 13:261-268. claimer. McDaniel, L.S., Scott, G., Kearney, J.F. and Briles, D.E., J. Exp. Med. vol. 160, Aug. 1984, pp. 386–397. Stover, C.K. et al., “New Use of BCG for Recombinant 21 Appl. No.: 08/468,985 Vaccines”, Nature, vol. 351, Jun. 6, 1991, pp. 456-460. 22 Filed: Jun. 6, 1995 DNA Sequencing, Chapter 13; A Laboratory Manual, 1989. McDaniel et al., 1989 Abstracts of the 89th Annual Meeting Related U.S. Application Data of the ASM, Abstract D-255. Rijn et al., “Growth Characteristics of Group A Streptococci 63 Continuation of application No. 08/319,795, Oct. 7, 1994, in a New Chemically Defined Medium”, Infection and which is a continuation-in-part of application No. 08/246, Immunity, Feb., 1980, pp. 444-448. 636, May 20, 1994, which is a continuation-in-part of R. Young and R. Davis, Proc. Natnl. Acad. Sci., 80, pp. application No. 08/048.896, Apr. 20, 1993, abandoned, which is a continuation-in-part of application No. 07/835, 1194-1198, 1983. 698, Feb. 12, 1992, abandoned, which is a continuation-in Ponger et al., Methods in Enzymology, 154: 450-473, 1987. part of application No. 07/656,773, Feb. 15, 1991, aban Abstracts of 89th Annual Meeting of the American Society doned. for Microbiology, p. 125, item D-257, May, 1989. Abstracts of 90th Annual Meeting of the American Society (51) Int. Cl. ............................................... A61K 39/09 for Microbiology, p. 98, Item D-106, May, 1990. 52 U.S. Cl. ..................................... 424/244.1; 424/237.1; Abstracts of 3d International ASM Conference on Strepto 530/350; 435/69.1; 435/320.1 coccal Genetics, p. 11, item 12, Jun., 1990. 58 Field of Search ..................................... 530/350, 820; Yother et al. Strep Genetics, p. 11, item 12, Jun. 1990. 424/237.1, 244.1; 435/320.1, 69.1 McDaniel et al., Abstracts of the 89th Annual Meeting of the ASM, Abstract D-255, 1989. 56) References Cited E. Harlow and d. Lane, “Antibodies, A Laboratory Manual”, See Chapter 13, pp. 511 to 551, 1988. U.S. PATENT DOCUMENTS Waltman et al., “Variation in the molecular weight of PspA 4,496,538 1/1985 Gordon ..................................... 424/92 (pneumococcal Surface protein A) among StreptococcuS 4,554,101 11/1985 Hopp .................................... 260/112.5 pneumoniae', Microbial Pathogenesis 1990, 8:61-69. 4,673,574 6/1987 Anderson .................................. 424/92 Waltman et al., Microbial Pathogenesis 1988, vol. 5: pp. 4,847,080 7/1989 Neurath et al. ........................... 424/89 159-167. 5,142,027 8/1992 Domen et al. .......................... 530/363 Cohen et al., Science 1994, 263:488–489. Engel et al., EMBO 1992, 11:4369–4378. OTHER PUBLICATIONS Cohen et al., 1990, Proteins 7:1-15. Fishetti et al., 1990, Molec. Micro. 4:1603–1605. Briles, D. and Yother, J., Sequence Analysis of pSpA Reveals Abstracts of the 90th Ann. Meeting of the ASM, 1990, that Pneumococcal Surface Protein A is a Complex Protein Yother et al. D-106. Containing at least Four Distinct Domains, Abstract, 3rd McDaniel et al. Infect. Immun. Jan. 91. 59(1):222-228. International ASM Conference on Streptococcal Genetics, Jun., 1990. Primary Examiner James C. Housel Briles, D. and Yother, J., Generation of truncated forms of ASSistant Examiner Jennifer Graser pneumococcal Surface protein A (PSpA) and DNA sequenc Attorney, Agent, or Firm Frommer Lawrence & Haug ing of pSpA, Abstract, 90th Annual Meeting of the American LLP, William S. Frommer; Thomas J. Kowalski Society for Microbiology, May, 1990. 57 ABSTRACT McDaniel et al. (IV) Infect Immun. 59:222-228, 1991. Yother, J., Briles, D.E. Structural and evolutionary relation Regions of the PSpA protein of the RX1 strain of Strepto ships in PSpA, a Surface protein of StreptococcuS pneumo COccuS pneumoniae have been identified as containing niae, as revealed by Sequence analysis. J. Bacteriol. protection-eliciting epitopes which are croSS-reactive with 174:601-609, 1992. PSpAS of other S. pneumoniae Strains and which is croSS Yother, J. Handsome, G.L., Briles, D.E. Truncated forms of protective. One region comprises the 68-amino acid PSpA that are Secreted from StreptococcuS pneumoniae and Sequence extending from amino acid residues 192 to 260 of their use in functional Studies and cloning of the pSpA gene. the RX1 PSpA Strain while another region comprises the J. Bacteriol. 174:610–618, 1992. C-terminal amino acid Sequence extending from amino acid McDaniel, L.S., Yother, J., Vijayakumar, M., McGarry, L., residues 293 to 588 of the RX1 PspA strain. Guild, W.R. and Briles, D.E., J. Exp. Med. vol. 165, Feb. 1987, pp. 381-394. 10 Claims, 7 Drawing Sheets U.S. Patent Dec. 7, 1999 Sheet 1 of 7 5,997,882 999 M10 3y9 ?00 91\\ 10uJ y sfi] |Z / / 99y993 91w1009y | |9 U.S. Patent Dec. 7, 1999 Sheet 2 of 7 5,997,882 919 |9A 102] 0.Jd U.S. Patent Dec. 7, 1999 Sheet 5 of 7 5,997,882 U.S. Patent Dec. 7, 1999 Sheet 6 of 7 5,997,882 O Xi 26 XiR35 XiR1224 XR16 15 XiR1iR15 26 Ox-helical 92 Xi84A Ar iR 1323" 260 3i.XiR 1325" Xi 288 proline rich 37O repeats (anchor) 588 COOH A/G 4 U.S. Patent Dec. 7, 1999 Sheet 7 of 7 5,997,882 4K 2K 3OK 46K 69K 375k 200k pKSDIO14 plN 5,997,882 1 2 EPITOPIC REGIONS OF PNEUMOCOCCAL In McDaniel et al (II), Microbial Pathogenesis SURFACE PROTEIN A 1:519-531, 1986, there are described studies on the charac terization of the PSpA. From the results of McDaniel (II), This application is a continuation of application Ser. No. McDaniel (III), J.Exp. Med 165:381-394, 1987, Waltman et 08/319,795, filed Oct. 7, 1994, which is a continuation-in al., Microb. Pathog. 8:61-69, 1990 and Crain et al., Infect. part application of Ser. No. 08/246,636 filed on May 20, Immun. 58:3293–3299, 1990, it was also apparent that the 1994, which is a continuation-in-part of application Ser. No. PspAs of different strains frequently exhibit considerable 08/048.896 filed Apr. 20, 1993 now abandoned, which is a diversity in terms of their epitopes, and apparent molecular weight. continuation-in-part of application Ser. No. 07/835,698 filed 1O Feb. 12, 1992 now abandoned and is a continuation-in-part In McDaniel et al (III), there is disclosed that immuniza of application Ser. No. 07/656,773 filed Feb. 15, 1991 which tion of X-linked immunodeficient (XID) mice with non is now abandoned. encapsulated pneumococci expressing PSpA, but not isogenic pneumococci lacking PSpA, protects mice from FIELD OF INVENTION 15 Subsequent fatal infection with pneumococci. This invention relates to recognition of epitopic regions of In McDanieletal (IV), Infect. Immun., 59:222–228, 1991, pneumococcal Surface protein A (PSpA), the major virulence there is described immunization of mice with a recombinant factor of StreptococcuS pneumoniae. full length fragment of PspA that is able to elicit protection BACKGROUND TO THE INVENTION against pneumococcal Strains of capsular types 6A and 3. In Crain et al., (Supra) there is described a rabbit antiserum StreptococcuS pneumoniae is an important cause of otitis that detects PSpA in 100% (n=95) of clinical and laboratory media, meningitis, bacteremia and pneumonia. Despite the isolates of strains of S. pneumoniae. When reacted with use of antibiotics and vaccines, the prevalence of pneumo Seven monoclonal antibodies to PSpA, fifty-Seven S. pneu coccal infections has declined little over the last twenty-five 25 moniae isolates exhibited thirty-one different patterns of yearS. reactivity. Accordingly, although a large number of It is generally accepted that immunity to StreptococcuS Serologically-different PSpAS exist, there are extensive pneumoniae can be mediated by Specific antibodies against croSS-reactions between PSpAS. the polysaccharide capsule of the pneumococcuS. However, The PSpA protein type is independent of capsular type. It neonates and young children fail to make an immune would seem that genetic mutation or exchange in the envi response against polysaccharide antigens and can have ronment has allowed for the development of a large pool of repeated infections involving the same capsular Serotype. Strains which are highly diverse with respect to capsule, One approach to immunizing infants against a number of PspA, and possibly other molecules with variable structures. encapsulated bacteria is to conjugate the capsular polysac 35 Variability of PSpA's from different strains also is evident in charide antigens to proteins to make them immunogenic. their molecular weights, which range from 67 to 99 kD. The This approach has been Successful, for example, with Hae observed differences are stably inherited and are not the mophilus influenzae b (see U.S.
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