As Choe et alhave demonstrated that sulfonation of tyrosine at amino acid 41 of the human Duffy antigen is essential for interaction with rPvDBPII, nDARC-Ig was expressed following cotransfection of mammalian cells with plasmids encoding nDARC-Ig and human tyrosyl protein sulfotransferase-2 [34]

As Choe et alhave demonstrated that sulfonation of tyrosine at amino acid 41 of the human Duffy antigen is essential for interaction with rPvDBPII, nDARC-Ig was expressed following cotransfection of mammalian cells with plasmids encoding nDARC-Ig and human tyrosyl protein sulfotransferase-2 [34]. horseradish peroxidase. Note that the refolded protein forms Rabbit Polyclonal to CD160 two bands that might represent slight variations in the way the protein refolds. This same pattern has been observed previously by Singh et al. [25]. (30 KB PDF) pmed.0040337.sg001.pdf (30K) GUID:?D788ED27-2F9F-46B1-846A-ACB8DB9D40CD Figure S2: ELISA to Determine Titer of Antibodies Rabbit antiserum was raised against the rPvDBPII Sal 1 variant. (A) ELISA titers of the rabbit antiserum to rPvDBPII Sal 1 and C variants. (B) Titers of affinity-purified human anti-PvDBPII Ab when attached to Sal 1 and C variants of rPvDBPII and to recombinant PvMSP119, a highly immunogenic antigen widely recognized by human anti-antibodies [51].(22 KB PDF) pmed.0040337.sg002.pdf (23K) GUID:?8070C2BD-982A-4791-B717-9B4732447091 Figure S3: COS Cell Binding Assay The COS cell binding assay Scopolamine reports the average of three independent experiments, each performed in triplicate on the rabbit anti-PvDBPII serum. The inoculated rabbit serum blocked COS7 cells expressing PvDBPII from forming rosettes in a dose-dependent fashion, leading to an indication that the PvDBPII was correctly folded Scopolamine and that antibodies directed against this protein were effective in interrupting the PvDBPIICDuffy interaction.(14 KB PDF) pmed.0040337.sg003.pdf (14K) GUID:?23ED1907-60D4-4AD4-9347-2E774364FE3A Abstract Background invasion requires interaction between the human Duffy antigen on the surface of erythrocytes and the Duffy binding protein (PvDBP) expressed by the parasite. Given that Duffy-negative individuals are resistant and that Duffy-negative heterozygotes show reduced susceptibility to blood-stage infection, we hypothesized that antibodies directed against region two of Duffy binding protein (PvDBPII) would inhibit invasion of human erythrocytes. Methods and Findings Using a recombinant region two of the Duffy binding protein (rPvDBPII), polyclonal antibodies were generated from immunized rabbits and affinity purified from the pooled sera of 14 merozoite. In vitro invasion assays, using blood isolates from individuals in the Mae Sot district of Thailand, showed that addition of rabbit anti-PvDBPII Ab or serum (antibodies against, or serum containing antibodies against, region two of the Duffy binding protein) (1:100) reduced the number of parasite invasions by up to 64%, while pooled PvDBPII antisera from invasion by up to 54%. Conclusions These results show, for what we believe to be the first time, that both rabbit and human antibodies directed against PvDBPII reduce invasion efficiency of wild isolated from infected patients, and suggest that a PvDBP-based vaccine may reduce human blood-stage infection. Editors’ Summary Background. Malaria is a parasitic infection transmitted to people through the bite of an infected mosquito. Four different parasites cause Scopolamine malariathe commonest and most widely distributed of these is are rarely fatal, but they cause debilitating chills and fevers that recur every other day if untreated. Like other malaria parasites, has a complex life cycle. Infected mosquitoes inject a form of the parasite known as sporozoites into people. The sporozoites replicate inside liver cells without causing any symptoms. Then, 8C9 d later, merozoites (another form of the parasite) are released from the liver cells and invade young red blood cells. Here, they replicate rapidly before bursting out and infecting more red blood cells. The characteristic symptoms of malaria are caused by this cyclical increase in the parasite burden. infections are usually treated with chloroquine, but patients must also take a second drug called primaquine. This drug kills hypnozoites, a form of the parasite that hibernates in the liver and that can cause a relapse many months after the initial bout of malaria. Why Was This Study Done? is becoming resistant to chloroquine and, although other antimalarial drugs still kill it, a vaccine that would limit the severity of infections by blocking its ability to invade red blood cells is urgently needed. The invasion of red blood cells by depends on an interaction between the Scopolamine Duffy antigen (a protein on the surface of human red blood cells) and the Duffy binding protein (PvDBP), which is expressed by merozoites. People who lack the Duffy antigen are resistant to blood-stage infections of merozoites. Then, the researchers showed that both types of antibody inhibited the binding of PvDBPII to Duffy antigen when the antigen was in solution and when it was present on human red blood cells. Finally, to test the ability of the antibodies to inhibit red blood cell invasion by the researchers established short-term cultures of the parasite from blood taken from infected adults living in Thailand. Addition of the rabbit or human antibodies to these cultures inhibited parasite invasion of.