Revista Iberoamericana de Micología

Revista Iberoamericana de Micología

Volume 36, Issue 4, October–December 2019, Pages 186-191
Revista Iberoamericana de Micología

Original article
Identifying molecularly defined antigens for a Histoplasma capsulatum-specific interferon gamma release assayIdentificación de antígenos definidos molecularmente para un ensayo de liberación de interferón-gamma específico para Histoplasma capsulatum

https://doi.org/10.1016/j.riam.2019.06.002Get rights and content

Abstract

Background

In a previous work we showed the feasibility of an interferon gamma release assay (IGRA) for detecting latent infection by Histoplasma capsulatum. While in that proof-of-concept study we used crude fungal extracts as antigens, the newest IGRAs developed for other infections are based on molecularly defined antigens, mostly on mixtures of immunogenic peptides.

Aims

To identify proteins in H. capsulatum that might serve as molecularly defined antigens for an IGRA test.

Methods

We surveyed the literature looking for known H. capsulatum-immunogenic proteins and assayed two of them as antigens in an IGRA test, in a study that involved 80 volunteers. Furthermore, we used several bioinformatics tools to identify specific H. capsulatum proteins and to analyze possible strategies for the design of H. capsulatum-specific immunogenic peptides.

Results

Seven H. capsulatum-immunogenic proteins were retrieved from the literature. IGRA tests using either the heat shock protein 60 or the M antigen showed high sensitivities but low specificities, most likely due to the high sequence similarity with the corresponding orthologs in other pathogenic microorganisms. We identified around 2000 H. capsulatum-specific proteins, most of which remain unannotated. Class II T-cell epitope predictions for a small number of these proteins showed a great variability among different alleles, prompting for a “brute force” approach for peptide design.

Conclusions

The H. capsulatum genome encodes a large number of distinctive proteins, which represent a valuable source of potential specific antigens for an IGRA test. Among them, the Cfp4 protein stands out as a very attractive candidate.

Resumen

Antecedentes

En un trabajo anterior mostramos la viabilidad de un ensayo de liberación de interferón-gamma (IGRA) para detectar la infección latente por Histoplasma capsulatum. En esa prueba de concepto utilizamos extractos crudos del hongo como antígenos; sin embargo, los IGRA de última generación desarrollados para otras infecciones se basan en antígenos definidos molecularmente, principalmente en mezclas de péptidos inmunogénicos.

Objetivos

Identificar proteínas de H. capsulatum que podrían servir como antígenos definidos molecularmente en una prueba IGRA.

Métodos

Examinamos la literatura en busca de proteínas inmunogénicas de H. capsulatum ya conocidas, y ensayamos dos de ellas como antígenos en una prueba IGRA, en un estudio donde participaron 80 voluntarios. Además, utilizamos varias herramientas bioinformáticas para identificar proteínas específicas de H. capsulatum y analizar posibles estrategias para el diseño de péptidos inmunogénicos específicos.

Resultados

Encontramos siete proteínas de H. capsulatum caracterizadas como inmunogénicas en la literatura. Las pruebas IGRA donde utilizamos la proteína de choque térmico 60 o el antígeno M, mostraron una alta sensibilidad, pero baja especificidad, debido probablemente a la alta similitud de secuencia con los ortólogos correspondientes en otros microorganismos patógenos. Identificamos unas 2000 proteínas específicas de H. capsulatum, la mayoría de las cuales permanecen sin anotar. Las predicciones de epítopos de células T de clase II realizadas para un pequeño número de estas proteínas mostraron una gran variabilidad entre los diferentes alelos, sustentando la aplicación de un enfoque de «fuerza bruta» en el diseño de estos péptidos.

Conclusiones

El genoma de H. capsulatum codifica una gran cantidad de proteínas específicas que representan una fuente valiosa de posibles antígenos para una prueba IGRA. Entre ellos, la proteína Cfp4 resulta un candidato muy atractivo.

Section snippets

Identification of H. capsulatum antigens

Two alternative strategies were explored in parallel to identify possible molecularly defined antigens that might be used in a H. capsulatum-specific IGRA test: (i) Searching for H. capsulatum proteins that have been previously characterized as immunogenic molecules; and (ii) Identification of specific H. capsulatum proteins, that is, proteins that do not have close orthologs in other related fungi. In both cases, the identified proteins might be used in the form of complete proteins or as

Immunogenic H. capsulatum proteins known from the literature

Table 1 lists seven proteins which have been evaluated before in multiple studies aiming to establish their role in the pathogenesis and immune response against H. capsulatum. Some of these proteins are part of the membrane and cell wall of the yeast phase, while others are secreted to the extracellular environment. Because of their proven immunogenicity, these proteins represent possible molecular antigens for a H. capsulatum-specific IGRA test.

IGRA tests using HSP60 or M antigen showed lack of specificity

We evaluated two of the H. capsulatum immunogenic

Conclusions

The development of an IGRA test for detecting latent infections by H. capsulatum should evolve toward the use of molecularly defined antigens, preferably immunogenic peptides. The use of known immunogenic H. capsulatum proteins having a high sequence similarity with their orthologs in other pathogenic microorganisms may result in a low test specificity, as demonstrated here for M and HSP60 antigens. Our bioinformatics analyses show that the H. capsulatum genome encodes a large number of

Funding

This work was supported by COLCIENCIAS (grant 221356933526) and the University of Antioquia (CODI-UdeA grant CIEMB-098-13).

Conflict of interest

The authors declare that they have no conflict of interest.

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