Polypeptides useful for diagnosis of Aspergillus fumigatus...

Details Polypeptides useful for diagnosis of Aspergillus fumigatus... Polypeptides useful for diagnosis of Aspergillus fumigatus...

1998-11-03

2001-07-17

Allen, Marianne P. (Department: 1631)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

15 to 23 amino acid residues in defined sequence

C530S327000, C530S328000, C530S329000

Reexamination Certificate

active

06262231

ABSTRACT:

FIELD OF INVENTION
The present invention relates to novel peptides of
Aspergillus fumigatus
having an amino acid sequence selected from the immunodominant region delimited by aa 6-22 of 18 kD allergen/antigen useful for immunodiagnosis.
The invention further relates to a method for using any of the peptide sequences of the present invention for the diagnosis of aspergillosis.
The invention also relates to a method for using the peptide sequence from aa 10-20 of the present invention for the diagnosis of aspergillosis.
The invention also relates to DNA sequences encoding for the peptides of the present invention.
The invention further relates to the DNA and RNA probes constructed on the basis of the peptide sequences of the present invention.
The invention also relates to recombinantly expressed peptides comprising the sequences of the peptides of the present invention.
The invention also relates to an immunodiagnostic kit using the peptides of the present invention for diagnosis of aspergillosis.
The invention further relates to a DNA based diagnostic kit using the DNA, cDNA or RNA sequences based on the sequences of the peptides of the present invention.
A field of use is the use of the said synthetic peptides for the preparation of a vaccine against aspergillosis.
A further field of use is the use of the said synthetic peptides for the detection of T-cell proliferation by in vitro tests.
A further field of use is use of said synthetic peptides for intradermal skin testing of aspergillosis.
BACKGROUND OF THE INVENTION
The fungui,
Aspergillus fumigatus
causes a wide spectrum of human and animal disorders such as allergic bronchopulmonary aspergillosis (ABPA), extrinsic allergic alveolitis, aspergilloma and invasive aspergillosis. Invasive form of aspergillosis is becoming increasingly important in immunosuppressed conditions due to environmental pollution, enhanced use of chemotherapeutic drugs and antibiotics etc. The most susceptible hosts are the immunocompromised patients, such as cases with organ transplant, leukemia or acquired immunodeficiency syndrome (AIDS).
Currently available tests for identification of this fungi is based on tedious, time consuming, less sensitive methods such as microscopy, cultures, electrophoresis and immunodiffusion. Many clinical features of aspergillosis are similar to tuberculosis and most of the aspergillosis patients are put on antituberculous therapy. The microscopy of the specimen for identification of Aspergillus hyphae is not easy under field conditions and specimens from the patients in the early stages of disease are often negative in the direct mounts. Further, the fungal culture generally takes 3-4 weeks and is expensive as a routine diagnostic measure. The widely used skin testing for Aspergillus allergic patients lacks sensitivity and specificity as the mixture of allergens used for testing is not well characterised and needs standardisation. The steroid therapy used for allergic patients and chemotherapy for invasive patients are more beneficial when employed in the early stages of the disease. Consequent to these factors, many investigators recommend that early diagnosis of aspergillosis should be considered as a priority area of research and development.
Peptides mimicking the epitopes in native antigens have been utilised in diagnosis as well as therapy of hepatitis, influenza, malaria, AIDS etc. Peptides based diagnosis of aspergillosis would be standardised and cost effective. Thus, the focus of research in aspergillosis pertaining to these aspects lies in the identification and purification of diagnostically relevant allergens and antigens of
A. fumigatus
and identification and synthesis of the epitopic peptides with sequences derived from the diagnostically relevant allergens and antigens.
PRIOR ART REFERENCES
Many investigators have identified protein allergens and antigens of
A. fumigatus
which have potential immunodiagnostic application. The N-terminal and internal amino acid sequences of many of these allergens/antigens have been published. The N-terminal amino acid sequences of some of these are presented in Table 1.
TABLE 1
A. fumigatus
protein allergens/antigens identified
by N-terminal amino acid sequences.
Investi-
gators
Antigens and N-terminus
Teshima
55 kDa: ATPHEPVFFSWDAGAVTSFP (SEQ ID NO:8)
et al,
1993

Kumar
65 kDa: AQNRQTLAKLLRYQSTKSG (SEQ ID NO:9)
et al,
1993

Moser
18 kDa: ATWTCINQQLNPKTNKWE (SEQ ID NO:10)
et al,
1992

Arruda
18 kDa: ATWTCINQQLNPKTNKWE (SEQ ID NO:10)
et al,
1992

Banerjee
34 kDa: SARDEAGLNEAVELARHAK (SEQ ID NO:11)
et al,
1996
However, none of the above allergens/antigens have been introduced as immunodiagnostic test products.
An alternative method of diagnosis of aspergillosis has been the use of polymerase chain reaction (PCR) and hybridisation. Various groups have identified and synthesized primers for genes specific to
A. fumigatus.
Our group also has developed a PCR based calorimetric diagnostic test specific for
A. fumigatus.
Gene based tests are highly sensitive and specific in comparison to immunodiagnosis. However, at present no gene based diagnostic kit for aspergillosis is available in the market as routine use of this test proves very expensive.
At present, an immunodiagnostic ELISA kit based on mixture of potent antigens has been formulated by the applicants. The present test is antibody based and thus is not useful for invasive patients.
The peptide based immunodiagnostic reagents are cost effective, homogeneous and more specific. Antigenic determinants of Asp fl have been synthesised by Kurup et al, 1995 but the diagnostic relevance of these peptides have not been indicated . As such, no peptide based immunodiagnostic kit is available in the market at present.
SUMMARY OF THE INVENTION
The present invention relates to novel peptide sequences from the immunodominant region of an 18 kD major allergen/antigen of
Aspergillus fumigatus
from aa 6-22, comprising aa 10-20, aa 6-20, aa 14-20, aa 10-22, aa 6-13 and the peptide sequence resulting from the modification by substitution and/or by addition and/or deletion of one or more amino acid altering specified properties. The peptides are useful in enzyme linked immunosorbent assay (ELISA) for the diagnosis of aspergillosis. They have lymphoproliferative as well as immunogenic properties and hence are potentially applicable in immunotherapy and immunoprophylaxis of aspergillosis. They are also able to stimulate the histamine release from sensitised mast cells of patients.
DETAILED DESCRIPTION
The present invention relates to epitopic peptides of an 18 kD major allergen/ antigen of
Aspergillus fumigatus
(strain 285, isolated from the sputum of an ABPA patient similar to the ATCC strain AF-102; ATCC42202).
A. fumigatus
causes allergic as well as invasive aspergillosis worldwide. Five epitopes were identified on the 18 kD major allergen/antigen in the immunodominant region from aa 6-22, comprising aa 10-20, aa 6-20, aa 14-20, aa 10-22, aa 6-13 these epitopic sequences were synthesised by solid phase method. The peptides were able to bind
A. fumigatus
specific antibodies in the sera of patients and hence, useful in enzyme linked immunosorbent assay (ELISA) for the diagnosis of aspergillosis. They have lymphoproliferative as well as immunogenic properties and hence are potentially applicable in immunotherapy and immunoprophylaxis of aspergillosis. They were also able to stimulate the histamine release from sensitised mast cells of patients.
The said peptides of the present invention are able to bind the
A. fumigatus
specific IgG and IgE antibodies in the sera of patients of aspergillosis. Further, they are able to raise significant amount of antibodies in the mice and these polyclonal antibodies can be used for diagnosis of invasive aspergillosis. These novel synthetic peptides are also able to stimulate histamine release from the sensitised mast cells of allergic bronchopulmonary aspergillosis patients and hence can replace crude mixture of allergens for skin testing.
The applicants have developed an enzyme li

Affiliated with

Also associated with

Rating

Polypeptides useful for diagnosis of Aspergillus fumigatus... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Polypeptides useful for diagnosis of Aspergillus fumigatus..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Polypeptides useful for diagnosis of Aspergillus fumigatus... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2537772