Treatment and detection of infection and disease associated with different fungal pathogens

Abstract

Described are immunogenic compositions comprising a non-naturally occurring pan-fungal Kex peptide, and methods of using such compositions for the treatment or prevention of infection and / or diseases associated with fungal pathogens (e.g., Pneumocystis, Aspergillus, Candida, or Cryptococcus) in the subject. Also provided are compositions and kits for detecting or quantifying the presence of antibodies directed against a non-naturally occurring pan-fungal Kex peptide in a subject.

Description

[0001] Cross References to Related Applications

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 796,120, filed January 24, 2019, the entire contents of which are incorporated herein by reference. Background technique

[0003] Pathogenic fungal organisms are ubiquitous in the environment and can cause global health threats. Pathogenic fungi are usually harmless to individuals with healthy and functioning immune systems, even after exposure (for example, through inhalation). However, individuals with weakened or compromised immune systems, or individuals with pre-existing medical conditions, such as individuals with lung disease or viral infections (such as HIV / AIDS infection), develop serious health problems after exposure and infection with fungal pathogens and There is a higher risk of adverse reactions.

[0004] Due to the severe impact of infections by fungal organisms on those who are in poor health and have weakened im...

Examples

preparation example Construction

[0162] The kit may also contain wash solutions or instructions for the preparation of wash solutions, where the combination of capture reagent and wash solution allows for the capture of anti-Kex antibodies on a solid support for subsequent detection by, for example, secondary antibodies, labeled reagent peptides, or mass spectrometry . In further embodiments, the kit may include instructions on suitable operating parameters in the form of a label or a separate insert (package insert). For example, the instructions may inform the consumer or user how to collect the sample, how to wash the anti-Kex peptide antibody and Kex peptide reagent complex after binding, how to interpret the results, etc. In yet another embodiment, the kit may include one or more containers containing appropriate positive and negative controls or control samples for use as standards for detection, calibration or normalization.

[0163] In another aspect, kits for treating or preventing an infection or d...

Embodiment 1

[0168] Example 1: Identification of the Kex1 pan-fungal consensus sequence

[0169] To identify the pan-fungal consensus sequence of Kex1, Kexin from Pneumocystis sp. (Accession No. EU918304.1) (isolated from macaque), Aspergillus fumigatus Kexin (Accession No. XM746441), Candida albicans Kexin (Accession No. AF022372) and Cryptococcus neoformans The KEX1 peptide sequence of Kexin (Accession No. XP572303.1) was subjected to multiple sequence alignment and analyzed using Clustal Omega (http: / / www.ebi.ac.uk / Tools / msa / clustalo / ) to analyze sequence identity and similarity sex( figure 1 ). figure 2 The resulting 90-mer Panfungal consensus KEX1 peptides (pantomycin 1 and pantomycin 2) shown in have about 97% and 69% amino acid sequence identity, respectively, to the amino acid sequence of Pneumocystis sp. KEX1 isolated from humans. The corresponding DNA sequence of the Panfungal 2 consensus Kex1 peptide was cloned into an E. coli expression vector, and the recombinant protein wa...

Embodiment 2

[0170] Embodiment 2: the production of recombinant Kexin protein

[0171] figure 1 The amino acid sequences of Aspergillus Kex, Cryptococcus Kex, Candida Kex, Panfungal Kex peptides 1 and 2 shown in were reverse translated using E. coli K-12 codon bias (www.ebi.ac.uk / Tools / st / emboss_backtranseq / ), and inserted into the expression vector pET-28b(+) using NcoI and BamHI restriction sites (GenScript). Pneumocystis sp. Kex1 was cloned using Genbank:EU918304.1 sequence. Each insert contains an additional GC followed by a CG at the 5' end of the Kex sequence to keep the Kex sequence insert in frame. The plasmid was transformed into E. coli BL21(DE3) cells and plated on LB agar supplemented with 40 μg / mL kanamycin to select transformed clones. All recombinant Kexin proteins were expressed and purified as described in Example 3 below.

[0001] Cross References to Related Applications

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 796,120, filed January 24, 2019, the entire contents of which are incorporated herein by reference. Background technique

[0003] Pathogenic fungal organisms are ubiquitous in the environment and can cause global health threats. Pathogenic fungi are usually harmless to individuals with healthy and functioning immune systems, even after exposure (for example, through inhalation). However, individuals with weakened or compromised immune systems, or individuals with pre-existing medical conditions, such as individuals with lung disease or viral infections (such as HIV / AIDS infection), develop serious health problems after exposure and infection with fungal pathogens and There is a higher risk of adverse reactions.

[0004] Due to the severe impact of infections by fungal organisms on those who are in poor health and have weakened im...

Examples

preparation example Construction

[0162] The kit may also contain wash solutions or instructions for the preparation of wash solutions, where the combination of capture reagent and wash solution allows for the capture of anti-Kex antibodies on a solid support for subsequent detection by, for example, secondary antibodies, labeled reagent peptides, or mass spectrometry . In further embodiments, the kit may include instructions on suitable operating parameters in the form of a label or a separate insert (package insert). For example, the instructions may inform the consumer or user how to collect the sample, how to wash the anti-Kex peptide antibody and Kex peptide reagent complex after binding, how to interpret the results, etc. In yet another embodiment, the kit may include one or more containers containing appropriate positive and negative controls or control samples for use as standards for detection, calibration or normalization.

[0163] In another aspect, kits for treating or preventing an infection or d...

Embodiment 1

[0168] Example 1: Identification of the Kex1 pan-fungal consensus sequence

[0169] To identify the pan-fungal consensus sequence of Kex1, Kexin from Pneumocystis sp. (Accession No. EU918304.1) (isolated from macaque), Aspergillus fumigatus Kexin (Accession No. XM746441), Candida albicans Kexin (Accession No. AF022372) and Cryptococcus neoformans The KEX1 peptide sequence of Kexin (Accession No. XP572303.1) was subjected to multiple sequence alignment and analyzed using Clustal Omega (http: / / www.ebi.ac.uk / Tools / msa / clustalo / ) to analyze sequence identity and similarity sex( figure 1 ). figure 2 The resulting 90-mer Panfungal consensus KEX1 peptides (pantomycin 1 and pantomycin 2) shown in have about 97% and 69% amino acid sequence identity, respectively, to the amino acid sequence of Pneumocystis sp. KEX1 isolated from humans. The corresponding DNA sequence of the Panfungal 2 consensus Kex1 peptide was cloned into an E. coli expression vector, and the recombinant protein wa...

Embodiment 2

[0170] Embodiment 2: the production of recombinant Kexin protein

[0171] figure 1 The amino acid sequences of Aspergillus Kex, Cryptococcus Kex, Candida Kex, Panfungal Kex peptides 1 and 2 shown in were reverse translated using E. coli K-12 codon bias (www.ebi.ac.uk / Tools / st / emboss_backtranseq / ), and inserted into the expression vector pET-28b(+) using NcoI and BamHI restriction sites (GenScript). Pneumocystis sp. Kex1 was cloned using Genbank:EU918304.1 sequence. Each insert contains an additional GC followed by a CG at the 5' end of the Kex sequence to keep the Kex sequence insert in frame. The plasmid was transformed into E. coli BL21(DE3) cells and plated on LB agar supplemented with 40 μg / mL kanamycin to select transformed clones. All recombinant Kexin proteins were expressed and purified as described in Example 3 below.

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