Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-02-09
2003-12-16
Benzion, Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S005000, C435S091100, C536S023100
Reexamination Certificate
active
06664053
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods for detecting
Pneumocystis carinii
infection in humans, specifically to such methods that involve polymerase chain reaction or other amplification of nucleic acid sequences that encode a
Pneumocystis carinii
sp. f. hominis protein.
BACKGROUND OF THE INVENTION
Pneumocystis carinii
is an important life threatening opportunistic pathogen of immunocompromised patients, especially those with human immunodeficiency virus (HIV) infection. Conventional diagnosis of
Pneumocystis carinii
pneumonia (PCP) involves analysis of a tissue sample or oropharyngeal secretion sample for the presence of a
P. carinii
organism through staining and microscopic examination. Sample acquisition techniques have included such invasive methods as transbronchial biopsy, percutanenous lung biopsy, or open lung biopsy. Each of these techniques is fraught with possible complications and requires significant time and expense. In the mid 1980's, bronchoalveolar lavage (BAL) was introduced as a less invasive, less expensive, and less complication-prone technique for acquiring samples to be used in PCP diagnosis (Ognibene et al. (1984)
Am. Rev. Respir. Dis
. 129:929-932). However BAL, coupled with bronchoscopy, still required special equipment and facilities, as well as the time of a physician and technician. Simpler still, it is now known that the Pneumocystis organism can also be detected in induced sputum samples (Bigby et al. (1986)
Am. Rev. Respir. Dis
. 133:515-518; Kovacs et al. (1988)
NEJM
318:589-593).
Advances also have occurred in the techniques used to detect the Pneumocystis organism in tissue and oropharyngeal secretion samples. Direct microscopic examination of clinical samples stained with, for instance, Giemsa stain or toluidine blue O, requires time-consuming sample preparation and subsequent examination by specially trained and experienced microscopy technicians (see, for instance, Bigby et al. (1986)
Am. Rev. Respir. Dis
. 133:515-518). This procedure has been somewhat simplified and rendered more amenable to mechanization through the use of monoclonal antibodies in detection of
P. carinii
antigens in clinical samples (Kovacs et al. (1988)
NEJM
318:589-593). A few groups have used oligonucleotide probes complementary to
P. carinii
nucleotide sequences to detect the organism through hybridization, as in U.S. Pat. No. 5,164,490 (the Santi patent).
Polymerase chain reaction (PCR) -mediated amplification of DNA or RNA-encoding sequences has been used to diagnose various diseases including leprosy (Santos et al. (1997)
J. Med. Microbiol
. 46:170-172) and PCP. This technique exhibits increased sensitivity over simple probe hybridization methods. Primers complementary to sequences encoding
P. carinii
mitochondrial or chromosomal ribosomal RNA (rRNA) have been used to amplify Pneumocystis-specific DNA sequence, as in Wakefield et al. (1990)
Mol. Biochem. Parasit
. 43:69-76; Wakefield et al. (1990)
Lancet
336:451453; Lipschik et al. (1992)
Lancet
340:203-206; WO 91/19005; and U.S. Pat. Nos. 5,519,127 (the Shah patent), 5,593,836 (the Niemiec patent) and 5,776,680 (the Leibowitz patent).
Other recent research advances relate to elucidating the molecular mechanisms involved in
P. carinii
infection. A great deal of interest has focused on the major surface glycoprotein (MSG; also called glycoprotein A) of
P. carinii
, because it is considered to be both a virulence factor and a target of host immune responses. MSG is the most abundant protein expressed on the surface of
P. carinii
, as assessed by Coomassie blue staining. It appears to play a critical role in the pathogenesis of pneumocystosis, possibly by acting as an attachment ligand to lung cells. MSG is also a target of both humoral and cellular immune responses by the host.
Multiple genes encode the MSG of rat-
P. carinii
, and different MSGs may be expressed in the lung of a rat infected with
P. carinii
(Angus et al. (1996)
J. Exp. Med
. 183:1229-1234; Kovacs et al. (1993)
J Biol. Chem
. 268:6034-6040). Similarly, multiple genes encode the MSG of
P. carinii
infecting ferrets and mice (Haidaris et al. (1998)
DNA Res
. 5:77-85; Haidaris et al. (1992)
J. Infect. Dis
. 166:1113-1123). Additional studies have shown that there is a single genomic site for expression of rat MSG variants (Edman et al. (1996)
DNA Cell Biol
. 15:989-999; Sunkin and Stringer (1996)
Mol. Microbiol
. 19:283-295; Wada and Nakamura (1996)
DNA Res
. 3:55-64; Wada et al. (1995)
J. Infect. Dis
. 171:1563-1568). These studies suggest that
P. carinii
has developed an elaborate system for antigenic variation, presumably to evade host defense mechanisms.
Molecular and immunological studies have clearly demonstrated that
P. carinii
isolated from different host species are distinct organisms, and may in fact be separate species (Gigliotti (1992)
J. Infect. Dis
. 165:329-336; Keely et al. (1994)
J. Eukaryot. Microbiol
. 41:94S; Kovacs et al. (1989)
J. Infect. Dis
. 159:60-70; Stringer (1993)
Infect. Agents Dis
. 2:109-117). There is a high level of variation among orthologous genes, including the MSG genes, isolated from different host-specific strains of the Pneumocystis. Hence, diagnosis of
P. carinii
infection in human patients ideally requires
P. carinii
sp. f. hominis (hereinafter “human-
P. carinii
”) derived reagents.
The cloning of human-
P. carinii
MSG genes has recently been reported (Garbe and Stringer (1994)
Infect. Immun
. 62:3092-3101; Stringer et al. (1993)
J. Eukaryot. Microbiol
. 40:821-826); however, only one full-length sequence was reported.
SUMMARY OF THE INVENTION
The inventors have discovered that human-
P. carinii
MSG is encoded for by a large, highly-conserved gene family, with a particularly conserved region of about 100 amino acids in the C-terminal region of the proteins. The have further discovered that direct detection or nucleic acid amplification (e.g., PCR amplification) of human-
P. carinii
MSG-encoding genes provides a particularly sensitive and specific technique for the detection of
P. carinii
, and the diagnosis of PCP.
This invention encompasses the purified novel human-
P. carinii
proteins represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14, and isolated nucleic acid molecules that encode these proteins. Specific nucleic acid molecules encompassed in this invention include those represented in SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4, SEQ ID NO: 5; SEQ ID NO: 6, SEQ ID NO: 7; SEQ ID NO: 15; and SEQ ID NO: 17. Also encompassed within this invention are the isolated nucleic acid sequences that encode the carboxy-terminal conserved about 100 amino acids of the disclosed human-
P. carinii
MSGs; these may be used for amplification or as probes. The sequences of these conserved nucleic acid molecule regions include residues 2794-3042 of HMSGp1 (SEQ ID NO: 1), 2758-3006 of HMSGp3 (SEQ ID NO: 3), 2845-3090 of HMSG11 (SEQ ID NO: 5), 2839-3084 of HMSG14 (SEQ ID NO: 7), 2836-3081 of HMSG32 (SEQ ID NO: 9), 2809-3054 of HMSG33 (SEQ ID NO: 11), 2821-3072 of HMSG35 (SEQ ID NO: 13), or 1-249 of HMSGp2 (SEQ ID NO: 15). In addition, this invention encompasses sequences with at least 70% sequence identity to these regions, and recombinant vectors comprising such nucleic acid molecules and conserved regions from within such nucleic acid molecules, as well as transgenic cells including such a recombinant vector.
Another aspect of this invention provides a method of detecting the presence of
Pneumocystis carinii
in a biological specimen, by amplifying with a nucleic acid amplification method (e.g., the polymerase chain reaction) a human-
P. carinii
nucleic acid sequence using two or more oligonucleotide primers derived from a human-
P. carinii
MSG protein encoding sequence, then determining whether an amplified sequence is present. In a preferred embodiment of this invention, the human-
P. carinii
nucleic acid sequence is a highly conserved region within an MSG-pr
Fischer Steven H.
Gill Vee J.
Huang Shengning
Kovacs Joseph A.
Masur Henry
Benzion Gary
Goldberg Jeanine
Klarquist & Sparkman, LLP
The United States of America as represented by the Department of
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