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Identification of mimotopes of Mycobacterium leprae as potential diagnostic reagents.

Alban SM, de Moura JF, Minozzo JC, Mira MT, Soccol VT - BMC Infect. Dis. (2013)

Bottom Line: An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients' sequelae.In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae.The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Engenharia de Bioprocessos e Biotecnologia, Universidade Federal do Paraná, Curitiba 81531-990, Brasil.

ABSTRACT

Background: An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients' sequelae. The currently used lepromin is not adequate for disease diagnosis and, so far, no antigen to be used in intradermoreaction has proved to be sensitive and specific for that purpose. Aiming at identifying new reagents to be used in skin tests, candidate antigens were investigated.

Methods: Random peptide phage display libraries were screened by using antibodies from leprosy patients in order to identify peptides as diagnostic reagents.

Results: Seven different phage clones were identified using purified antibodies pooled from sera of leprosy patients. When the clones were tested with serum samples by ELISA, three of them, 5A, 6A and 1B, allowed detecting a larger number of leprosy patients when compared to controls. The corresponding peptides expressed by selected phage clones were chemically synthesized. A pilot study was undertaken to assess the use of peptides in skin tests. The intradermal challenge with peptides in animals previously sensitized with Mycobacterium leprae induced a delayed-type hypersensitivity with peptide 5A (2/5) and peptide 1B (1/5). In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae.

Conclusions: The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.

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Related in: MedlinePlus

Delayed-type hypersensitivity response induced by peptides in guinea-pigs. Guinea-pigs were sensitized with 200 μg M. leprae in Freund’s incomplete adjuvant and - after 30 days - were inoculated intradermally with peptides and controls. Each group contained five animals. The result in 48 hours after application of 10 μg peptides, Mistsuda lepromin (M) with 4 × 106 bacilli and 10 μg soluble extract of M. leprae (ML) is indicated. The peptide pool contain equimolar amounts of the three peptides. The diameter of the reaction corresponds to the average of horizontal and vertical diameters of the erythema. 0.9% NaCl (C) was used as negative control. * The animal responded after 72 hours. ** The animal responded after 21 days.
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Figure 4: Delayed-type hypersensitivity response induced by peptides in guinea-pigs. Guinea-pigs were sensitized with 200 μg M. leprae in Freund’s incomplete adjuvant and - after 30 days - were inoculated intradermally with peptides and controls. Each group contained five animals. The result in 48 hours after application of 10 μg peptides, Mistsuda lepromin (M) with 4 × 106 bacilli and 10 μg soluble extract of M. leprae (ML) is indicated. The peptide pool contain equimolar amounts of the three peptides. The diameter of the reaction corresponds to the average of horizontal and vertical diameters of the erythema. 0.9% NaCl (C) was used as negative control. * The animal responded after 72 hours. ** The animal responded after 21 days.

Mentions: Since a humoral response depends on T helper cell response, the peptides identified were assessed regarding their ability to induce a delayed-type hypersensitivity response in guinea-pigs sensitized with M. leprae. The use of guinea-pigs constitutes the animal model used in delayed-type hypersensitivity tests with M. leprae antigens, according to description presented previously[17,20-22]. The intradermal challenge with peptides in animals previously sensitized with M. leprae induced delayed-type hypersensitivity. The maximum reading was observed in 48 hours and, therefore, that time was considered in the description of the results. No reactions were detected in animals sensitized with adjuvant as well as in non-sensitized animals. In the group sensitized with M. leprae, a DTH reaction was observed with peptide 5A (2/5), peptide 1B (1/5), Mitsuda lepromin (3/5), and with M. leprae (4/5) (Figure 4). For peptide 1B, the skin reaction was observed for both 2 and 10 μg doses with similar sizes - 16.5 mm and 15.5 mm respectively - whereas for peptide 5A, only the 10 μg dose generated a response. The two animals that did not respond to Mitsuda lepromin in 48 hours produced a response after 72 hours (induration) or 21 days (induration) after the application of the antigen. The detection of response with Mitsuda lepromin and the soluble extract of M. leprae indicate that the animals were sensitized. The skin reactions observed were erythematous; only one of the animals immunized with M. leprae, besides the erythema, also developed a papule as a result of applying peptide 5A.


Identification of mimotopes of Mycobacterium leprae as potential diagnostic reagents.

Alban SM, de Moura JF, Minozzo JC, Mira MT, Soccol VT - BMC Infect. Dis. (2013)

Delayed-type hypersensitivity response induced by peptides in guinea-pigs. Guinea-pigs were sensitized with 200 μg M. leprae in Freund’s incomplete adjuvant and - after 30 days - were inoculated intradermally with peptides and controls. Each group contained five animals. The result in 48 hours after application of 10 μg peptides, Mistsuda lepromin (M) with 4 × 106 bacilli and 10 μg soluble extract of M. leprae (ML) is indicated. The peptide pool contain equimolar amounts of the three peptides. The diameter of the reaction corresponds to the average of horizontal and vertical diameters of the erythema. 0.9% NaCl (C) was used as negative control. * The animal responded after 72 hours. ** The animal responded after 21 days.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585472&req=5

Figure 4: Delayed-type hypersensitivity response induced by peptides in guinea-pigs. Guinea-pigs were sensitized with 200 μg M. leprae in Freund’s incomplete adjuvant and - after 30 days - were inoculated intradermally with peptides and controls. Each group contained five animals. The result in 48 hours after application of 10 μg peptides, Mistsuda lepromin (M) with 4 × 106 bacilli and 10 μg soluble extract of M. leprae (ML) is indicated. The peptide pool contain equimolar amounts of the three peptides. The diameter of the reaction corresponds to the average of horizontal and vertical diameters of the erythema. 0.9% NaCl (C) was used as negative control. * The animal responded after 72 hours. ** The animal responded after 21 days.
Mentions: Since a humoral response depends on T helper cell response, the peptides identified were assessed regarding their ability to induce a delayed-type hypersensitivity response in guinea-pigs sensitized with M. leprae. The use of guinea-pigs constitutes the animal model used in delayed-type hypersensitivity tests with M. leprae antigens, according to description presented previously[17,20-22]. The intradermal challenge with peptides in animals previously sensitized with M. leprae induced delayed-type hypersensitivity. The maximum reading was observed in 48 hours and, therefore, that time was considered in the description of the results. No reactions were detected in animals sensitized with adjuvant as well as in non-sensitized animals. In the group sensitized with M. leprae, a DTH reaction was observed with peptide 5A (2/5), peptide 1B (1/5), Mitsuda lepromin (3/5), and with M. leprae (4/5) (Figure 4). For peptide 1B, the skin reaction was observed for both 2 and 10 μg doses with similar sizes - 16.5 mm and 15.5 mm respectively - whereas for peptide 5A, only the 10 μg dose generated a response. The two animals that did not respond to Mitsuda lepromin in 48 hours produced a response after 72 hours (induration) or 21 days (induration) after the application of the antigen. The detection of response with Mitsuda lepromin and the soluble extract of M. leprae indicate that the animals were sensitized. The skin reactions observed were erythematous; only one of the animals immunized with M. leprae, besides the erythema, also developed a papule as a result of applying peptide 5A.

Bottom Line: An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients' sequelae.In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae.The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Engenharia de Bioprocessos e Biotecnologia, Universidade Federal do Paraná, Curitiba 81531-990, Brasil.

ABSTRACT

Background: An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients' sequelae. The currently used lepromin is not adequate for disease diagnosis and, so far, no antigen to be used in intradermoreaction has proved to be sensitive and specific for that purpose. Aiming at identifying new reagents to be used in skin tests, candidate antigens were investigated.

Methods: Random peptide phage display libraries were screened by using antibodies from leprosy patients in order to identify peptides as diagnostic reagents.

Results: Seven different phage clones were identified using purified antibodies pooled from sera of leprosy patients. When the clones were tested with serum samples by ELISA, three of them, 5A, 6A and 1B, allowed detecting a larger number of leprosy patients when compared to controls. The corresponding peptides expressed by selected phage clones were chemically synthesized. A pilot study was undertaken to assess the use of peptides in skin tests. The intradermal challenge with peptides in animals previously sensitized with Mycobacterium leprae induced a delayed-type hypersensitivity with peptide 5A (2/5) and peptide 1B (1/5). In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae.

Conclusions: The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.

Show MeSH
Related in: MedlinePlus