Plant Expression of Hydrophobin Fused K39 Antigen for Visceral Leishmaniasis Immunodiagnosis.

Bruno B. Silva,Lívia É. C. Marques,Arnaldo S. Bezerra,Maurício F. van Tilburg,Eduarda N. F. N. Santos,Eridan O. P. Tramontina Florean,Maria Izabel F. Guedes,Lucelina S. Araújo

doi:10.3389/fpls.2021.674015

Abstract

Visceral leishmaniasis is a Neglected Tropical Disease of high mortality caused by the protozoan Leishmania infantum. Its transmission cycle is complex, and it has in the domestic dog its main reservoir. The diagnostic tests currently used rely on prokaryotic systems’ proteins, but their low sensitivity increases the disease’s burden. The plant transient expression of recombinant proteins allows the production of complex antigens. However, this system has limited competitiveness against the bacterial production of purified antigens. Thus, we have shown that the L. infantum K39 antigen’s fusion to a hydrophobin allows its production for diagnostic tests without the need for intensive purification. The sera of naturally infected dogs specifically detect the semi-purified rK39-HFBI protein. The test validation against a panel of 158 clinical samples demonstrates the platform’s viability, resulting in sensitivity and specificity of 90.7 and 97.5%, respectively. Thus, the use of semi-purified antigens fused to hydrophobins can become the standard platform for large-scale antigens production to expand diagnostic tests for other human and veterinary diseases worldwide.

Highlights

Visceral leishmaniasis is a Neglected Tropical Disease that annually afflicts 300,000 people worldwide, resulting in the death of 20,000 of them (World Health Organization, 2018; Ruiz-Postigo et al, 2020)
To produce the rK39 antigen fused to a hydrophobin, we infiltrated the leaves of 6–8 weeks old N. benthamiana with an A. tumefaciens LBA4404 culture transformed with a binary plasmid containing the construct rK39-HFBI
For rK39-HFBI, the protein expression dynamics’ evaluation required normalization by the leaf area instead of the fresh plant weight, since necrosis of the leaf tissue was initiated at 6 d.p.i. and progressed until the material was infeasible for analysis at 10 d.p.i

Summary

Introduction

Visceral leishmaniasis is a Neglected Tropical Disease that annually afflicts 300,000 people worldwide, resulting in the death of 20,000 of them (World Health Organization, 2018; Ruiz-Postigo et al, 2020). In the Americas, visceral leishmaniasis is a zoonotic disease, with the transmission cycle involving the domestic dog (reservoir), the protozoan Leishmania infantum (etiological agent) and the sandfly Lutzomyia longipalpis (primary vector; Burza et al, 2018). Brazil accounts for 96% of all visceral leishmaniasis cases reported in Latin America (WHO-PAHO, 2018), highlighting the need for new strategies of prophylaxis, diagnosis and treatment for the control of the disease. The serological diagnosis of visceral leishmaniasis relies on the recombinant K39 antigen expressed in Escherichia coli (Faria and de Andrade, 2012). The K39 protein is predominant in the amastigote stage of L. infantum, and it belongs to the kinesin superfamily. These proteins comprise a class of eukaryotic motor proteins involved in microtubule-based movements. The number of antibodies that bind to these antigens is proportional to their number of repeats (Goto et al, 2010)

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