Serological diagnosis of pneumocystosis: production of a synthetic recombinant antigen for immunodetection of Pneumocystis jirovecii

Abstract

Diagnosis of Pneumocystis pneumonia (PcP) relies on the detection of P. jirovecii in respiratory specimens obtained by invasive techniques. Thus, the development of a serological test is urgently needed as it will allow the diagnosis of PcP using blood, an inexpensive and non-invasive specimen. This study aims to combine the production of a multi-epitope synthetic recombinant antigen (RSA) and an ELISA test for detection of anti-P. jirovecii antibodies, in order to develop a new approach for PcP diagnosis. The RSA was selected and designed based on the study of the immunogenicity of the carboxyl-terminal domain of the major surface glycoprotein. This antigen was purified and used as an antigenic tool in an ELISA technique for detection of Ig, IgG and IgM antibodies anti-P. jirovecii (patent-pending no. PT109078). Serum specimens from 88 patients previously categorized in distinct clinical subgroups and 17 blood donors, were analysed. The IgM anti-P. jirovecii levels were statistically increased in patients with PcP (p = 0.001) and the ELISA IgM anti-P. jirovecii test presented a sensitivity of 100% and a specificity of 80.8%, when associated with the clinical diagnosis criteria. This innovative approach, provides good insights about what can be done in the future serum testing for PcP diagnosis.

Development of a Gold Nanoparticle-Based Lateral-Flow Immunoassay for Pneumocystis Pneumonia Serological Diagnosis at Point-of-Care

“…Recently, studies showed that a technology based on synthetic amino acid sequences, designed to hold more than one reactive region of the selected antigens, could enhance the immunological diagnosis of Toxoplasma gondii (Dai et al, 2012(Dai et al, , 2013. Therefore, in our previous study, this research group designed a recombinant synthetic antigen (RSA) with three antigenic regions of the Msg protein, in order to standardize and enhance the detection of reactive antibodies against P. jirovecii (Tomás et al, 2016). In this single antigenic tool, several proven reactive and conserved fragments of the Msg were used, improving its immunogenic power and consequently its application as an anti-P. jirovecii antibody detection tool (Tomás et al, 2016).…”

“…Therefore, in our previous study, this research group designed a recombinant synthetic antigen (RSA) with three antigenic regions of the Msg protein, in order to standardize and enhance the detection of reactive antibodies against P. jirovecii (Tomás et al, 2016). In this single antigenic tool, several proven reactive and conserved fragments of the Msg were used, improving its immunogenic power and consequently its application as an anti-P. jirovecii antibody detection tool (Tomás et al, 2016). In the present study, this RSA was used in combination with a new RSA, produced based on the immunogenic behavior of P. jirovecii Kex1 protein.…”

“…Yet, reports of protection against acquisition of infection by passive transfer of immune sera in mice (Gigliotti et al, 2002) and by vaccination in immunosuppressed nonhuman primates (Kling and Norris, 2016), triggers interest in serum antibodies as serological biomarkers of the disease. In addition, the suggestion that the IgM isotype has a predominant role in shaping the earliest steps in recognition and clearance of Pneumocystis infection both in mice (Rapaka et al, 2010) and in humans (Djawe et al, 2010;Tomás et al, 2016), not only support the role of antibodies in disease protection, but also highlights the idea that a serological test for PcP diagnosis is viable.…”

“…The Msg RSA was designed as previously described (Tomás et al, 2016) and Kex1 RSA followed the same procedure, studying the immunogenicity of the P. jirovecii's KEX1 longest gene sequences available (GenBank: AY130996.1 and AY127566.1) (Kutty and Kovacs, 2003). The putative reactive epitopes were selected using bioinformatic tools to analyze electrochemical properties, secondary structure prediction, polarity, relative position to the membrane and hydrophobicity profile of the specific polypeptides at the online software ExPASy -ProtScale and CBS -TMHMM -version 2.0 (Dai et al, 2012(Dai et al, , 2013Tomás et al, 2016). Only specific and conserved regions of Pneumocystis KEX1 genes, presenting high similarity with the sequences previously reported in GenBank for Pneumocystis' Kex1 protein were considered for the final selection of the RSA composition, through the NCBI blast tool.…”

“…As P. jirovecii's major surface glycoproteins (Msg) are characteristic of this microorganism and highly immunogenic, containing both B and T cell protective epitopes (Stringer and Keely, 2001), they are the obvious candidate to study serological responses. In fact, promising studies using recombinant antigens of this protein and antibody immunodetection techniques, have shown that patients with PcP or previous episodes of PcP present higher serum levels of anti-P. jirovecii antibodies than patients without P. jirovecii infection or without previous PcP events (Daly et al, 2004;Djawe et al, 2010;Gingo et al, 2011;Blount et al, 2012;Tomás et al, 2016). However, as Msg presents antigenic variation during infection as an evasion mechanism (Kling and Norris, 2016;Hauser, 2019), other antigenic candidates began to been explored.…”