Detection of Plasmodium falciparum,P. vivax,P. ovale,and P. malariae Merozoite Surface Protein 1-p19 Antibodies in Human Malaria Patients and Experimentally Infected Nonhuman Primates

Approximately 3.2 billion people live in areas where malaria is endemic, and WHO estimates that 350 to 500 million malaria cases occur each year worldwide. This high prevalence, and the high frequency of international travel, creates significant risk for the exportation of malaria to countries where malaria is not endemic and for the introduction of malaria organisms into the blood supply. Since all four human infectious Plasmodium species have been transmitted by blood transfusion, we sought to develop an enzyme-linked immunosorbent assay (ELISA) capable of detecting antibodies elicited by infection with any of these species. The merozoite surface protein 1 (MSP1), a P. falciparum and P. vivax vaccine candidate with a well-characterized immune response, was selected for use in the assay. The MSP1 genes from P. ovale and P. malariae were cloned and sequenced (L. Birkenmeyer, A. S. Muerhoff, G. Dawson, and S. M. Desai, Am. J. Trop. Med. Hyg. 82:996-1003, 2010), and the carboxyl-terminal p19 regions of all four species were expressed in Escherichia coli. Performance results from individual p19 ELISAs were compared to those of a commercial test (Lab 21 Healthcare Malaria enzyme immunoassay [EIA]). The commercial ELISA detected all malaria patients with P. falciparum or P. vivax infections, as did the corresponding species-specific p19 ELISAs. However, the commercial ELISA detected antibodies in 0/2 and 5/8 individuals with P. malariae and P. ovale infections, respectively, while the p19 assays detected 100% of individuals with confirmed P. malariae or P. ovale infections. In experimentally infected nonhuman primates, the use of MSP1-p19 antigens from all four species resulted in the detection of antibodies within 2 to 10 weeks postinfection. Use of MSP1-p19 antigens from all four Plasmodium species in a single immunoassay would provide significantly improved efficacy compared to existing tests.More than 3.2 billion people in the world today live in areas where malaria is endemic. The World Health Organization estimates that more than 350 to 500 million malaria clinical disease episodes occur each year worldwide, with more than 1 million deaths occurring annually in sub-Saharan Africa, mostly among children under the age of 5 years (50). The combination of high disease prevalence and high frequency of international travel creates a significant risk for the exportation of malaria to countries where the disease is nonendemic. This risk is accompanied by the potential for introduction of malaria-causing organisms into the blood supplies used for transfusions. All four principal species of Plasmodium that infect humans have been transmitted via blood transfusion in the United States (36), France (4), the United Kingdom (23), and Switzerland (19). This has resulted in the implementation of donor deferral policies in many countries that restrict blood donation by those with a history of recent travel to or emigration from regions of endemicity and by those with recent cases of clinical malaria. Recent publications indicate that the prevalence of Plasmodium knowlesi, a pathogen of simian origin, in human populations in Southeast Asia (11, 12), Singapore (37), the Philippines (31), and Thailand (21) is much higher than previously believed. However, P. knowlesi malaria appears to be a zoonotic disease and to our knowledge has been not implicated in cases of transfusion-transmitted malaria in humans.The effectiveness of donor deferral programs has previously been questioned (29), and there is concern that many donors are needlessly deferred, since the rates of imported malaria are much lower than the rates of travel to areas of endemicity (17, 35). To prevent erosion of qualified donor populations, some countries have implemented antibody screening such that only individuals who are known to have been exposed to organisms causing malaria are subject to deferral of donations rather than all donors who have traveled to or lived in regions where malaria is endemic. Commercial antibody enzyme-linked immunosorbent assays (ELISAs) are currently in use (in the United Kingdom, France, and Australia), and reinstatement of questionnaire-deferred donors is being discussed in Canada and the United States (16, 24, 42). In these cases, potential donors are tested for antibodies to Plasmodium-derived antigens within several months of deferral; when the tested individuals show negative antibody results, donation is allowed.Antibodies to asexual malaria parasites (i.e., merozoites) appear within days to weeks after the invasion of erythrocytes and can persist for months or even years (14, 49). Historically, antibodies to parasite antigens have been detected using the immunofluorescence assay (IFA). This assay is not particularly sensitive or specific and is labor-intensive, requiring careful preparation of reagents. Commercially available ELISAs have been developed that use recombinant antigens or P. falciparum whole-organism lysates for detection of immunoglobulins (IgG and/or IgM, IgA) in human serum or plasma (Lab 21 Healthcare Laboratories, United Kingdom; Cellabs, Australia; DiaMed AG, Switzerland; LG Chemical Inc., Iksan, South Korea; Green Cross, Inc., Youngin, South Korea [Genedia Malaria Ab Rapid]; and Standard Diagnostics, Suwon, South Korea). These assays are typically easier to perform and exhibit higher throughput and better sensitivity and specificity than IFA (25, 42, 47), though this is not always the case (32). Some ELISAs may be better than others for detection of antibodies against all four Plasmodium species that cause malaria in humans (44). However, none of the available commercial assays currently include P. ovale- or P. malariae-derived antigens. Because these organisms have been implicated in transfusion-transmitted malaria (TTM), it would be advantageous to include antigens from these organisms in an antibody detection assay.Antigens used in some commercial ELISAs for the capture of antibodies have included vaccine candidates, since their ability to elicit antibody responses in animals and human vaccine recipients has been predetermined and naturally occurring antibodies are measured prior to vaccination. Examples of such antigens include circumsporozoite protein (CSP), apical membrane antigen 1 (AMA-1), merozoite surface protein 1 (MSP1), and, in particular, a 19-kDa C-terminal fragment of MSP1 (MSP1-p19) (22, 25, 40). Plasmodium falciparum MSP1 has been extensively studied and was one of the very earliest vaccine candidates; it elicits a protective antibody response against severe malaria, and the presence of MSP1 antibodies correlates with protective immunity (45). MSP1 is expressed as an ∼200-kDa precursor molecule linked by a glycosyl phosphatidylinositol anchor to the merozoite surface membrane. MSP1 is processed into a complex of polypeptides on the merozoite surface, including N-terminal and central regions of 82, 30, and 38 kDa, as well as the C-terminal region of 42 kDa. At the time of invasion of red blood cells, MSP1-p42 is further processed by proteolytic cleavage into a 33-kDa fragment (MSP1-p33), which is shed with the rest of the complex, and a C-terminal 19-kDa fragment (MSP1-p19). Only the C-terminal MSP1-p19 fragment remains anchored on the merozoite surface and is carried into parasitized red blood cells (RBC) (10). In monkeys, immunization with recombinant P. falciparum MSP1-p42 and P. falciparum MSP1-p19 has been shown to elicit various degrees of protection against P. falciparum challenge (15, 26). MSP1-p19 proteins from both P. falciparum and P. vivax have been proposed as vaccine candidates (18, 41, 48).By analogy to P. falciparum and P. vivax findings, one would predict that the MSP1 genes of P. ovale and P. malariae would be useful as reagents for vaccination or antibody detection. We recently cloned and expressed the MSP1-p19 proteins of P. malariae and P. ovale as recombinant antigens in Escherichia coli (2). We report here the independent evaluation of these proteins as reagents for antibody detection using sera from human malaria patients and experimentally infected nonhuman primates. In addition, a prototype immunoassay combining MSP1-p19 antigens from all four Plasmodium species was evaluated and its performance compared to a commercially available antibody test.