Modified immunogenic pneumolysin compositions as vaccines

a technology of immunogenic pneumolysin and composition, applied in the field of vaccines, can solve the problems of respiratory burst, phagocytic function of polymorphonuclear leukocytes, and ineffective vaccines in all populations, and achieve the effect of inducing protective immunity

Inactive Publication Date: 2005-03-31
MINETTI CONCEICAO +5
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, PLY may be the principal cause of hearing loss and cochlear damage in a guinea pig model of pneumococcal meningitis (Winter et al.
Unfortunately, these vaccines are not effective in all populations, especially those of Asia.
It has also been demonstrated that the respiratory burst, chemotactic, and phagocytic functions of polymorphonuclear leukocytes, all of which are critically important for removing invading pneumococci, are severely compromised in the presence of pneumolysin.
However, anomalous electrophoretic mobility indicates that the protein is incorrectly folded.

Method used

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  • Modified immunogenic pneumolysin compositions as vaccines
  • Modified immunogenic pneumolysin compositions as vaccines
  • Modified immunogenic pneumolysin compositions as vaccines

Examples

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example 1

Expression of Pneumolysin.

[0135]E. coli strain BL21 (DE3) Δompa transformed with pET-17b or pET-24a containing the desired gene was grown with moderate aeration at 30° C. in LB supplemented with 0.4% glucose and 100 μg / ml of carbenicillin (for pET-17b constructs) or 50 μg / ml of kanamycin (for pET-24a constructs). When the OD600 reached 0.6, IPTG was added to a final concentration of 0.4 mM (for pET-17b constructs) of 1 mM (for pET-24a constructs) and the cells were allowed to incubate for another 2 h for screening, or 5 h for larger scale production. To assay for pneumolysin levels, 1.5 ml aliquots were removed prior to induction and at various time points after induction and examined by SDS-PAGE.

example 2

Cloning of the Pneumolysin Gene for Streptococcus pneumoniae serotype 14.

[0136] Genomic DNA was isolated from approximately 0.5 g Streptococcus pneumoniae serotype 14 using the method described above. This DNA served as the template for two pneumolysin-specific oligonucleotides in a standard PCR reaction. These oligonucleotides were designed to be complementary to the 5′ and 3′ flanking regions of the pneumolysin gene from S. pneumoniae serotype 2 and to contain XbaI restriction sites to facilitate the cloning of the fragment if desired. The sequence of the forward oligonucleotide was 5′ AAC CTT GAT TGA TCT AGA TAA GGT ATT TAT GTT GG 3′ and the reverse oligonucleotide had the sequence 5′ TCT TTT TGT CTC TAG AAT TCT CCT CTC CTA GTC 3′. The PCR reaction conditions were as follows: 200 ng S. pneumoniae type 14 genomic DNA, the two oligonucleotide primers described above at 1 μM of each, 200 μM of each dNTP, PCR reaction buffer (10 mM Tris HCl, 50 mM KCl, pH 8.3), 1.5 mM MgCl3, and 2.5...

example 3

Expression of the pneumolysin gene in E. coli.

[0137] Plasmids capable of expressing the mature pneumolysin protein were constructed by amplifying DNA containing the full-length pneumolysin gene (pST20, pST85, or type 14 genomic DNA) with nested oligonucleotides designed to isolate the pneumolysin coding region. The forward oligonucleotide was designed to contain a NdeI site and would install a start codon at the 5′ end of the coding region. This primer had the sequence 5′ TAT TAG GAG GAG CAT ATG GCA AAT AAA GCA GTA AAT G 3′. The reverse oligonucleotide was designed to contain an XhoI site and had the sequence 5′ GGC CTC TTT TTG TCT CGA GCA TTC TCC TCT CCT AGT C 3′. This strategy allowed the cloning of the fragment encoding mature pneumolysin into the NdeI and XhoI sites of either the pET-17b or pET-24a. Standard PCR was conducted using a template containing the entire pneumolysin gene (type 1, 2 & 14) and the two oligonucleotides described above. This PCR reaction yielded a 1.6 kb ...

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Abstract

This invention relates to modified pneumolysin polypeptides that retain the immunogenic nature of pneumolysin but have reduced or undetectable hemolytic activity compared to native pneumolysin. The invention also provides a method for generating novel pneumolysin variants with these desired characteristic properties. The invention also provides immunogenic compositions useful as pharmaceutical compositions including vaccines in which non-toxic, modified pneumolysin is used to stimulate protective immunity against Streptococcus pneumoniae. The vaccines may be compositions in which the modified pneumolysin is conjugated to bacterial polysaccharides or may be carried on an attenuated viral vector. In addition, the invention also provides a method of using the non-toxic, modified pneumolysin toxoid in order to stimulate antibodies against Streptococcus pneumoniae in a treated individual which are then isolated and transferred to a second individual, thereby conferring protection against Streptococcus pneumoniae in the second individual.

Description

FIELD OF THE INVENTION [0001] This invention relates to the field of vaccines, and in particular, methods for the production of modified forms of pneumolysin and their use in producing compositions for the immunization of mammals against infections caused by bacteria including Streptococcus pneumoniae. BACKGROUND OF THE INVENTION [0002]Streptococcus pneumoniae is the major cause of bacterial pneumonia, bacteremia, meningitis, and otitis media (Baltimore et al. in Bacterial infections of humans: Epidemiology and control Evans and Brachman eds, Plenum Press, New York, 1989 pp.525-546; Schuchat et al. N. Engl. J. Med. 1997, 337, 970-976). Even with appropriate antibiotic therapy, pneumococcal infections have been estimated to result in as many as 40,000 deaths a year in the United States (Fedson et al. Archives of Internal Medicine 1994, 154, 2531-2535; Fiebach et al. Archives of Internal Medicine 1994, 154, 2545-2557). In addition, pneumococci have gained increased resistance to penic...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/195C12N15/09C07K14/315C12N1/19C12N1/21C12N5/10
CPCA61K39/092A61K2039/6037Y10S424/831Y10S530/825C07K14/3156C07K14/00
Inventor MINETTI, CONCEICAOMICHON, FRANCISPULLEN, JEFFREYPOLVINO-BODNAR, MARYLIANG, SHU-MEITAI, JOSEPH
Owner MINETTI CONCEICAO
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