First characterization of Plasmodium vivax liver stage antigen (PvLSA) using synthetic peptides
© Goo et al.; licensee BioMed Central Ltd. 2014
Received: 15 July 2013
Accepted: 3 February 2014
Published: 12 February 2014
Plasmodium vivax is the most widespread human malaria in tropical and subtropical countries, including the Republic of Korea. Vivax malaria is characterized by hypnozoite relapse and long latency infection by the retained liver stage of P. vivax, and somewhat surprisingly, little is known of the liver stage antigens of this parasite. Here, we report for the first time the characterization of a liver stage antigen of P. vivax (PvLSA).
Five peptides located inside PvLSA were synthesized, and specific anti-sera to the respective peptides were used to localize PvLSA on P. vivax parasites in human liver cells by immunofluorescence. Western blotting and enzyme-linked immunosorbent assay were performed using the five peptides and sera collected from vivax malaria patients and from normal healthy controls.
PvLSA was localized on P. vivax parasites in human liver cells. Vivax malaria-infected patients were detected using the five peptides by western blotting. Furthermore, the peptides reacted with the sera of vivax malaria patients.
These results suggest that PvLSA may function during the liver stage of P. vivax.
KeywordsPlasmodium vivax Liver stage antigen Peptides
Plasmodium vivax is the most widespread human malaria, and afflicts several hundred million people annually. It is endemic to tropical and subtropical countries of the Americas, Africa, and Asia, including the Republic of Korea (ROK)[1–3]. Unlike P. falciparum, P. vivax is characterized by hypnozoite relapse in the liver. After being bitten by a P. vivax-infected mosquito, sporozoites enter hepatocytes, where most develop into schizonts that result in primary illness. However, some remain as hypnozoites, which can become active months or even years later, and cause relapse after resolution of the primary illness. Several factors have been suggested to lead to hypnozoite development, for example, a cold ambient temperature, the number of infecting sporozoites, the specific strain of the mosquito vector or P. vivax[4–6]. However, the mechanisms responsible for hypnozoite development and their activation are not known.
A vaccine and a diagnostic method based on antigens specific to the liver stage of P. vivax are needed in order to control vivax malaria, since asymptomatic carriers in latency contribute to disease transmission. In falciparum malaria, a recombinant anti-sporozoite subunit vaccine (RTS,S/AS01) targeting circumsporozoite protein (CSP) has shown best performance among vaccines developed to date, though Phase III trials are ongoing. In addition, the detection of human carriers in the latent stage caused by hypnozoites is important in many countries, including the ROK, where the control strategy for vivax malaria is moving from intervention toward elimination. Therefore, an understanding of molecules specific for the liver stage could help overcome the challenge posed by vivax malaria in the setting of disease elimination.
In P. falciparum, liver stage antigen-3 (LSA-3) is a novel antigen expressed at the pre-erythrocytic stage. A number of studies have demonstrated the potential of LSA-3 as a vaccine and serodiagnosis candidate. B- and T-cell epitopes have been characterized in LSA-3, and LSA-3 antigenicity has been demonstrated in several immuno-epidemiological studies conducted in P. falciparum malaria-exposed populations. Moreover, an enzyme-linked immunosorbent assay (ELISA) based on recombinant LSA-3 has been developed as a serodiagnostic test for P. falciparum in Myanmar. On the other hand, little is known about the molecular characteristics of the liver stage of P. vivax, and the majority of studies conducted, since Garnham identified the pre-erythrocytic stage of P. vivax in human liver in 1947, focused on the biology of hypnozoites.
Synthetic peptides derived from antigens of Plasmodium spp. provide practical advantages for vaccine development, evaluations of antigenicity[14, 15], and surveys of immunologic profiles in malaria-exposed populations. Furthermore, ELISA tests developed for peptides of some promising antigens now have improved performances.
Therefore, we synthesized peptides that span all liver stage antigens of P. vivax (PvLSA), and evaluated the antigenicities of these peptides by Western blotting. Finally, the efficacies of ELISA for these peptides were determined based on its ability to detect blood samples from vivax malaria patients.
The study was performed in the ROK and in Thailand, and was approved by the ethics committee of the Korean National Institute of Health (Approval number: 2009-01CON-01-4R). An approval form was used to obtain written informed consent from each participant. In addition, all participants provided permission for the sampling of 5 ml of blood.
Blood samples, which were collected in EDTA tubes, were obtained from 65 patients diagnosed with vivax malaria at local health centers (Gang-wha, Paju, Gimpo) from March to August. Microscopic examinations of Giemsa-stained thick and thin blood films were used to confirm diagnoses. Samples were also obtained from 10 asymptomatic and aparasitemic healthy volunteers confirmed as being P. vivax negative by microscopic examination and nested-PCR.
Selection and synthesis of antigenic peptides on Plasmodium vivax liver stage antigen
Plasmodium vivax sporozoite preparation
P. vivax sporozoites were prepared at the Armed Forces Research Institute of Medical Sciences (AFRIMS; Thailand), as described previously[20, 21]. Briefly, sporozoites were collected from the salivary glands of Anopheles dirus (Bangkok Colony) mosquitoes fed the blood of vivax malaria patients. Sporozoites, in an aseptic solution containing 200 U/ml penicillin and 200 μg/ml streptomycin, were centrifuged and counted. Subsequently, they were inoculated into HC-04 cells (a human hepatocyte cell line) that had been cultured in complete medium (MEM: Ham’s F12 Gibco BRL, 1:1 v/v) supplemented with 10% fetal bovine serum (Gibco BRL), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C for 48 h. HC-04 cells were harvested on days 1, 2, and 3 after sporozoite inoculation. 3After washing, cells were spread as a monolayer on cytospin slides (ThermoShandon, USA).
Immunofluorescence assay (IFA)
To determine whether PvLSA was expressed in liver stage parasites, we performed an immunofluorescence assay (IFA) using HC-04 cells and specific anti-sera to the five peptides at the Armed Forces Research Institute of Medical Sciences (AFRIMS; Thailand). Anti-sera specific to the respective peptides (P1-5) were purchased from Peptron Inc. (ROK). The slides prepared as described above were first fixed with cold acetone for IFA. Next, anti-P1, P2, P3, P4, and P5 rabbit sera, and the monoclonal antibody of the circumsporozoite protein type VK210 were used at concentrations of 10 μg/ml for IFA staining. Secondary antibodies were fluoresced using isothiocyanate-conjugated anti-rabbit and human IgG (Invitrogen, USA), and fluorescence was visualized by confocal microscopy (Leica, Germany). An anti-serum to the circumsporozoite antigen expressed in P. vivax parasites during the early liver stage was used as a positive control.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting
Briefly, the five ovalbumin-conjugated peptides were separated by SDS-PAGE and stained with Coomassie Blue. Separated peptide fractions were electroblotted onto Immobilon-P Transfer membranes (Millipore, USA), which were then blocked with 5% skim milk (Wako, Japan). Subsequently, membranes were probed overnight with vivax malaria sera samples diluted in 5% skim milk. Bound antibodies were reacted with horseradish peroxidase-conjugated secondary antibodies and detected using the West-Q Chemiluminescent Substrate Kit (GenDEPOT, USA).
Enzyme-linked immunosorbent assay (ELISA)
The 60 blood samples from vivax malaria patients and 10 samples from healthy controls were subjected to ELISA as previously described with modifications. Peptides without ovalbumin (P1, 2, 3, and 5) (500 ng) were coated onto 96-well microplates (Nunc, Denmark) overnight at 4°C, and then incubated with respective blood samples at a dilution of 1:200 (peptide 4, which showed weak reaction in a localization study was excluded). Second antibody binding was detected using horseradish peroxidase-conjugated anti-human IgG (Bethyl Laboratories, Inc., USA) (1:5000) and TMV (Sigma-Aldrich, USA). Optical densities were measured at 450 nm. The cut-off value of each peptide was calculated by adding 3 times the standard deviation of 10 blood samples from healthy people to the mean OD value. Samples with an OD value higher than the appropriate cut-off value were considered vivax malaria positive.
Results and discussion
Positive rate of vivax malaria by ELISAs with peptides (P1, 2, 3 and 5) spanning on PvLSA
No. of positive sample (%)
No. of negative sample (%)
Although PvLSA and liver stage antigens of P. falciparum (PfLSA-1 and PfLSA-3) are antigenic proteins expressed by malaria parasites in hepatocytes, PvLSA and PfLSAs are distinct at the genomic sequence level. PvLSA is homologous with the liver stage antigen of P. cynomolgi (PCYB_092710, PlasmDB), but not with two liver stage antigens of P. falciparum. In addition, PvLSA does not have the specific repeat and non-repeat domains of the liver stage antigens of P. falciparum. Although PvLSA and the liver stage antigens of falciparum malaria (PfLSA-1 and PfLSA-3) have different functions in the liver stages of vivax and falciparum malaria.
Studies on molecules expressed by malarial parasites during the liver stage, such as, on liver stage antigen, could help identify the mechanisms responsible for the long-term survival of vivax parasites in the human liver and of latent infection. In addition, serodiagnostic methods and vaccines based on synthetic peptides have been recently developed for parasitic diseases and other pathogenic infections[16, 24]. Therefore, we suggest further studies on the five peptides are warranted to provide more insight on the liver stage.
We report for the first time, the characterization of a liver stage antigen of P. vivax using five synthesized peptides located on PvLSA. Specific anti-sera produced using the respective peptides were found to react with P. vivax parasites in human liver cells. Furthermore, peptides specifically reacted with sera from vivax malaria patients by ELISA. Based on these results, further studies would provide more insight on the liver stage of vivax malaria.
This study was supported by an intramural grant from the Korea National Institute of Health (#2011-N54003-00).
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