Chlamydia proteins and their uses

Details Chlamydia proteins and their uses Chlamydia proteins and their uses

2000-10-16

2004-06-08

Swartz, Rodney P. (Department: 1645)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Bacterium or component thereof or substance produced by said...

C424S009100, C424S009200, C424S184100, C424S185100, C424S190100, C424S234100, C424S278100, C530S300000, C530S350000

Reexamination Certificate

active

06746676

ABSTRACT:

I. FIELD OF THE INVENTION
The present invention relates to the detection of Chlamydia and to the diagnosis, treatment and prevention of Chlamydia infections in animals.
II. BACKGROUND
Chlamydiae are obligate intracellular bacterial pathogens with a unique biphasic life cycle. They appear as two distinct cellular types, a small dense cell or elementary body (EB) that is enclosed in a rigid bacterial cell wall, and a larger metabolically active reticulate body (RB). The EB is resistant to physical disruption and is infectious, whereas the RB is more fragile and only exists inside cells. The Chlamydia life cycle begins with the attachment of the EB form to the host cell which is followed by endocytosis into a nascent vacuole, also called an “inclusion membrane”. After EB attachment and entry, replication of the EB form produces RB forms that continue to grow within the vacuole. By 72 hour post-infection, this growth phase is terminated when the RBs condense, and reorganize back to EBs. The lysis of the host cell results in release of EBs to infect new host cells. The difficulties in working with Chlamydiae center on the obligate intracellular requirement for growth and the fact that no adequate genetic engineering methods have been developed for this organism.
The genus Chlamydia includes two species that are primarily associated with human disease:
C. trachomatis
and
C. pneumoniae. C. trachomatis
causes trachoma, an eye disease that is the leading cause of preventable infectious blindness worldwide with an estimated 500 million cases of active trachoma worldwide.
C. trachomatis
also causes a sexually transmitted chlamydial disease which is very common worldwide.
C. trachomatis
also causes lymphogranuloma venereum, a debilitating systemic disease characterized by lymphatic gland swelling. The most serious sequelae of chlamydial genital infections of females include salpingitis, pelvic inflammatory disease, and ectopic pregnancy. In the U.S. alone, it is estimated that over 4 million new sexually transmitted
C. trachomatis
infections occurred in 1990, leading to over four billion dollars in direct and indirect medical expenses. The World Health Organization estimates that 89 million new cases of genital Chlamydia occurred worldwide in 1995 (Peeling and Brunham, 1996).
C. pneumoniae
causes respiratory diseases including so called walking pneumonia, a low-grade disease such that the infected person frequently fails to obtain treatment and remains in the community as an active, infectious carrier.
C. pneumoniae
is currently of interest because of its strong epidemiological association with coronary artery disease, and there is also some evidence to link it with multiple sclerosis.
Of the other disease-causing species of Chlamydia,
Chlamydia psittaci
and
Chlamydia pecorum
are primarily pathogens of wild and domestic animals, but these species may infect humans accidentally.
C. psittaci
is acquired through respiratory droplet infection and is considered an occupational health hazard for bird fanciers and poultry workers.
There is tremendous interest in the identification of candidate antigens for protection against chlamydial disease. While a prior infection with
C. trachomatis
will protect against a subsequent challenge by the same strain, indicating a protective component that stimulates the host immune response, most serious chlamydial diseases are exacerbated by an overaggressive anti-chlamydial immune response. Antigens recognized in the context of an infection appear to elicit a protective response whereas immunization with purified, killed (EB form) Chlamydia results in an immunopathological response. Therefore for the purposes of vaccine development, one needs to find epitopes that confer protection, but do not contribute to pathology. It is an object of this invention to provide Chlamydia polypeptides for use as vaccines that induce a protective immune response without inducing the pathological response caused by the antigens associated with the EB form of Chlamydia. Such immunostimulatory peptides will be useful in the treatment, as well as in the diagnosis, detection and prevention of Chlamydial infections.
III. SUMMARY OF THE INVENTION
The present invention includes the use of Chlamydia proteins that show enhanced expression in the reticulate body (RB) stage relative to the elementary body (EB) stage of the Chlamydia life cycle. These proteins are not present at detectable levels in the EB form using current immunological techniques and are thus said to be “infection-specific.” Certain of these infection-specific proteins are found in the inclusion membrane of the infected cell, and so have been termed “Inc” proteins. These include the IncA, IncB, and IncC proteins of Chlamydia as described in the present disclosure. The genes that encode the IncA, IncB and IncC proteins are referred to as incA, incB and incC respectively. Other proteins of Chlamydia described herein have also been shown by the inventors to be infection-specific, but are not known to be incorporated into the inclusion membrane; these include the p242, TroA, and TroB proteins. The TroA and TroB proteins have been so named because they resemble the Tro proteins of
Treponema pallidum
, which are thought to form part of an ABC transport system.
The inventors have shown that the infection-specific Chlamydia proteins of the disclosure are recognized by convalescent antisera (i.e., antisera taken from an animal that has recovered from a Chlamydia infection) but are not recognized by antisera against the killed EB form of Chlamydia. Thus, the proteins are expressed only during active chlamydial infection and are therefore useful as protective antigens. These infection-specific proteins may be used to confer a protective immune response without inducing a pathological effect. Additionally, immuno-fluorescence microscopy and immunoblotting with antisera demonstrated that the infection-specific proteins are present in Chlamydia-infected HeLa cells, but are undetectable in purified EBs and absent in uninfected HeLa cells.
Immunofluorescense microscopy reveals that IncA, IncB and IncC are localized to the inclusion membrane of infected HeLa cells. Reverse-transcription polymerase chain reactions (RT-PCR), northern hybridization data, and restriction analysis revealed that the incB and incC genes are closely linked and transcribed in an operon. RT-PCR, restriction analysis and sequential Southern hybridizations of incA then incC to the same filter provided evidence that incA is separated from the incB and incC operon by about 110 kb. The
C. trachomatis
Tro genes are not closely linked with the p242 gene.
The present invention includes the nucleotide and amino acid sequences for certain infection-specific proteins from Chlamydia. These proteins are p242, TroA, and TroB from
C. trachomatis
, and the IncB, and IncC proteins from
C. psittaci
. The scope of the invention includes fragments of these proteins that may be used in a vaccine preparation or that may be used in a method of detecting Chlamydia antibodies. Such fragments may be, for example, 5, 10, 15, 20, 25, or 30 contiguous amino acids in length. They may even encompass the entire protein.
More specifically, the present invention encompasses the purified infection-specific proteins having amino acid sequences as shown in SEQ ID NOS: 2, 4, 6, 10, and 12, amino acid sequences that differ from such sequences by one or more conservative amino acid substitutions, and amino acid sequences that show at least 75% sequence identity with such amino acid sequences.
Then invention also includes isolated nucleic acid molecules that encode a protein as described in the above paragraph, including isolated nucleic acid molecules with nucleotide sequences as shown in SEQ ID NOS: 1,3, 5, 9, and 11.
The present invention also includes a vaccine or immunostimulatory preparation directed against the reticulate body (RB) form of Chlamydia comprising one or more purified infection-specific peptides (or portions or fragments thereof, or peptides showing sequence

Affiliated with

Also associated with

Rating

Chlamydia proteins and their uses does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Chlamydia proteins and their uses, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Chlamydia proteins and their uses will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3341937