Novel monoclonal antibodies against Plasmodium falciparum histidine-rich protein 2: development and application in rapid diagnostic tests of malaria in hyperendemic regions of China and Myanmar

Kang et al. BMC Microbiology Novel monoclonal antibodies against Plasmodium falciparum histidine-rich protein 2: development and application in rapid diagnostic tests of malaria in hyperendemic regions of China and Myanmar Keren Kang 0 1 Emmanuel E Dzakah 0 3 Wenmei Li 0 Mingquan Xie 1 Xiaochun Luo 1 Hui Liu 2 0 National Engineering Laboratory of Point-of-Care Tests, Guangzhou Wondfo Biotech Co. Ltd , Guangzhou 510663 , China 1 School of Bioscience and Bioengineering, South China University of Technology , Guangzhou 510006 , China 2 Yunnan Provincial Center for Parasitic Diseases Control and Prevention (NIPD) , Yunnan 650000 , China 3 Department of Molecular Biology and Biotechnology, School of Biological Sciences, University of Cape Coast , Cape Coast , Ghana Background: Malaria presents a considerable threat to public health. Histidine-rich protein 2 (HRP 2) is the major protein released into human blood upon infection by Plasmodium falciparum. In this study, we aimed to evaluate the immunogenicity of HRP 2 exon II and the efficacy of novel monoclonal antibodies (mAbs) against HRP 2 for Point-of-Care Test (POCT). Methods: The recombinant protein was expressed in soluble form in E. coli and used to immunize mice for mAb production. Two IgG1 mAbs (1A5 and 1C10) with high affinity, specificity and sensitivity for both native and recombinant HRP 2 were selected after fusion of mouse spleen with myeloma cells. The affinity constant of 1A5 and 1C10 were 7.15 and 4.91 10-7 L/mol, respectively. Subsequently, an immunochromatograhic assay was used for screening of clinical samples in endemic regions of China and Myanmar. Results: The immunochromatographic test retrospectively showed an overall sensitivity of 99.07%, and specificity of 100%. Sensitivity at parasite densities < 200, 200-2000, and > 2000 parasites/L was 87.5, 98.7, and 100%, respectively. Conclusions: These results suggest that HRP 2 exon II contains immunogenic sites similar to those of the native antigen and can be used for the development of mAbs suitable for malaria diagnosis in endemic communities. Malaria; Histidine-rich protein 2 exon II; Monoclonal antibodies; Point-of-care test; Immunochromatographic test - Background Malaria, a disease caused by Plasmodium species, is one of the oldest and largest health challenges affecting 40% of the world population [1]. There were an estimated 627, 000 malaria deaths in 2012, including 91% in Africa, mainly caused by P. falciparum infection, the most lethal malarial plasmodia responsible for the cerebral form of the disease. Approximately 86% malaria deaths are children under 5 years of age [2-4]. These estimates rank malaria as one of the top three killers among infectious diseases in the world. Although Plasmodium falciparum prevalence rates in most parts of China and Myanmar have been brought under control, very high transmission rates still remain in certain regional communities [5]. Due to the economic boom in China, cross border transmission has resulted in increasing malaria incidence in recent years. Current economic globalization trends coupled with marked movement of people have accelerated the incidence of Plasmodium related cases with increased antimalarial drug resistance in Southeast Asia, including China [5]. The Yunnan Province alone counts over ten million cases of malaria among border and immigration officers, an indication of the prevalence of malaria at both sides of the border. Continuous migration of the population in border areas makes it extremely difficult to implement malaria control programs. Available data in recent years have also shown imported cases of malaria in Henan, Hebei, Fujian, Chongqing, Shanghai, Jiangxi and among others [6]. Inaccurate and ineffective diagnosis of P. falciparum in these regions has resulted in drug resistant species, pointing to the need for improved diagnosis and monitoring of the disease. The total eradication of malaria is one of the urgent aims of the United Nations Millennium Development Goals. The methods recommended by WHO for P. falciparum diagnosis include microscopic examination, immunological tests, and PCR methods [7-9]. Since the launch of WHOs T3 initiative in 2012, all malaria-endemic and donor countries should ensure that every suspected case of malaria is tested and treated, [10] requiring increased developent of rapid diagnostic tests (RDTs). Hence the use of fast, accurate, and easy on-site detection methods and reagents as monitoring medical tools for early diagnosis and treatment of malaria in regions of high transmission and prevalence is particularly important. Several detection antibodies against different Plasmodium antigens have been described, of which Plasmodium falciparum specific histidine-rich protein 2 (HRP 2), and Plasmodium lactate dehydrogenase (pLDH) and aldolase are common to all four Plasmodium species [11]. The PfHRP 2 gene is located on chromosome 8 of the parasite and comprised of both exons I and II, encoding a 309-amino acid protein. Sequence variations among the different strains range between 800 to 1300 base pairs. The PfHRP exon II alone encodes 287 amino acids composed of 34.5% histidine and 35 repeats of the tripeptide His-His-Ala sequences. HRP 2 is released upon rupture of parasitized erythrocytes at late-stage [12] and is capable of reversing the tightly balanced activities of anticoagulant factors that maintain homeostasis [13]. The most convenient technique for clinical diagnosis of malaria is the use of rapid diagnostic test kits, which depends on the use of mAbs against the HRP 2 antigen. These tests are particularly important since they can be used in field diagnostics (point-of-care test, POCT) to screen large populations without the requirement of trained laboratory personnel or equipment. Several tests targeting HRP 2 are available, with various specificities, sensitivities, and temperature tolerances, illustrating the difficulties and challenges facing current RDTs [14]. The difficulties associated with RDTs include genetic variability in the HRP 2 gene and the persistence of antigens in the bloodstream following the elimination of parasites [14]. For instance, ParaSight F, a sensitive, specific, simple, and fast dipstick assay, uses mAb IgG1, a subclass of IgG [15,16]. However, HRP 2-based RDTs have given false-positive results and there has been some debate as to the cause of these false positives [14]. Therefore, the development and production of mAbs with high affinity, specificity and sensitivity will help facilitate its use as POCT. In this study, we aimed to express the HRP 2 exon II antigen retaining the essential epitopes that can serve as immunogens for use in developing mAbs against HRP 2 for POCT immunochromatographic assay, according to guiding principles formulated by the WHO [10]. The results of assays were compared with those of microscopic examination known as gold standard for clinically screening of malaria samples in (...truncated)