Recombinant 60-kDa heat shock protein from Paracoccidioides brasiliensis: is it a good antigen for serological diagnosis of paracoccidioidomycosis?

Abstract

Paracoccidioides brasiliensis and P. lutzii are fungi that cause paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in South America. For serological diagnosis, although 43-kDa glycoprotein (gp43) is regarded as highly specific for PCM, the occurrence of false negative reactions in sera from patients infected with P. lutzii suggests that preparation with only one antigen is not recommended. Heat shock proteins are feasible alternatives as a second antigen because they are often highly immunogenic. In this study, we evaluated the usefulness of recombinant 60-kDa heat shock protein from P. brasiliensis (rPbHsp60) for the serological diagnosis of PCM. Using western blotting assay, we observed that 77.3% of the sera from PCM patients were positive to rPbHsp60, with 90.9% positivity to recombinant gp43 (rgp43). More importantly, sera from healthy subjects had 27% positivity to rPbHsp60 and none to rgp43. When rPbHsp60 was used in ELISA, we did not observe significant differences between the reactions with sera from PCM patients and healthy subjects, while the difference was clearly evident when the antigen was rgp43. Furthermore, rPbHsp60 was recognized by sera from patients with histoplasmosis, aspergillosis, sporotrichosis or tuberculosis in an ELISA test. These results show that rPbHsp60 is not a good antigen for PCM diagnosis.

Updates in Paracoccidioides Biology and Genetic Advances in Fungus Manipulation

“…In addition, mice immunized with P. brasiliensis Hsp60 recombinant protein showed a protective immune response and a significant reduction in fungal burden after infection with P. brasiliensis [163]. Although the presence of antibodies against Hsp60 appears to be insufficient to induce protection, since recombinant Hsp was recognized by sera from PCM patients [164,165], mice previously treated with monoclonal antibodies (mAbs) generated against Hsp60 from Histoplasma capsulatum showed a decrease in pulmonary fungal load after challenge with P. lutzii. The protective effects induced by mAbs were partially due to an increase in phagocytosis exhibited by P. lutzii yeast cells opsonized with these mAbs [166].…”

Metanálisis de pruebas inmunológicas para el diagnóstico de la infección por Paracoccidioides, 1972-2017

“…En alusión a la especificidad, la mejor se encontró en el estudio de Ortiz con 90% (IC95% 55-100) 34 . La proteína HSP60 fue otro de los antígenos evaluados; sin embargo, su desempeño no alcanzó el 80% en ninguno de los parámetros (Perón 2017) 33 .…”

“…Ortiz et al reportaron una especificidad del 87,5%, la cual disminuye al comparar la reactividad con sueros heterólogos a 58,7%; así como una sensibilidad del 73,4%, sugiriendo que la proteína p27 puede representar un sólo epítope y por lo tanto ser reconocido sólo por algunos pacientes con PCM 34 . Por otro lado, la HSP60 pertenece a la familia de proteínas de choque térmico (HSP) y es altamente inmunogénica, sin embargo su desempeño en los estudios incluidos varió significativamente pese a la utilización de la misma técnica (Western blotting ), Cunha et al, indica una sensibilidad de 97,3% y especificidad de 92,5% 35 , mientras que Perón et al reporta 77,3% de sensibilidad y 73% de especificidad con evidencia de reacciones cruzadas en pacientes con histoplasmosis, probablemente originado por la homología de la proteína con HSP presente en Histoplasma capsulatum y en sujetos sanos por la presencia de anticuerpo anti-HSP 33 .…”

Applications of western blot technique: From bench to bedside

“…Western blot has enough sensitivity to be included among routine laboratory assays as a safe method for PCM diagnosis. 83,84 Since western blot has been developed, it has been able to adopt by biology labs and turns to one of the most important techniques in research and clinical applications. Despite some technical limitation related to performance and sensitivity, further advancements can improve sensitivity and reproducibility of the WB.…”