As a result, out of the 325 selected proteins, the ASC of 3 proteins showed a significant positive correlation with GIA activity (Table 1, Table S2). measurements, antibody reactivity against 1-Methyladenine 3 proteins was positively associated with GIA activity. Since anti-LSA3-C responses showed the strongest correlation with GIA activity, this protein was investigated further. Anti-LSA3-C-specific antibody purified from Malian adult plasmas showed GIA activity, and expression of LSA3 in blood-stage parasites was confirmed by western blotting. Taken together, we identified LSA3 as a novel blood-stage vaccine candidate, and we propose that this system will be useful for future vaccine candidate discovery. WHO estimated 214 million cases and 438,000 deaths from malaria in 2015, and was responsible for much of this morbidity and mortality1. The emergence of drug-resistant parasites and insecticide-resistant mosquitoes has greatly hampered malaria control and has accelerated development of new approaches to support malaria eradication and elimination efforts2,3. Moreover, classic studies showing that passive transfer of -globulin isolated from adults who lived in a malaria endemic area dramatically reduced parasitemias and alleviated the symptoms in malaria-infected children have pointed to the role of antibodies in protective immune responses4. However, the targets of this naturally acquired immunity are not fully comprehended. Malaria vaccine candidates under preclinical and clinical development are currently limited (WHO rainbow table: http://www.who.int/vaccine_research/links/Rainbow/en/index.html), and several blood-stage vaccines, which have reached to clinical trials, failed to show efficacy in field studies or in controlled human malaria infection models5,6. Therefore, more candidates for malaria vaccines for further blood-stage vaccine development are needed. When attempted, the screening for novel candidates has been hampered in part by troubles in expressing plasmodial proteins in heterologous 1-Methyladenine expression systems. Correct protein conformations are essential to induce protective immunity against malaria7, and it is well acknowledged that recombinant plasmodial proteins cannot usually elicit functional antibodies due to the lack of proper conformation8. There are several preceding studies to detect a broader range of antigen-specific immune responses using protein microarrays9,10,11, and an growth inhibitory antibodies in animals as judged by functional assays with parasites, such as the growth inhibition assay (GIA)8,12. The results indicate that this recombinant proteins expressed by WGCFS retain (at least in part) critical functional epitopes. Purified IgGs from residents in malaria endemic areas have shown GIA activities13,14. To identify novel blood-stage malaria vaccine candidates, in this study 1,827proteins were expressed using WGCFS, and 51 purified IgGs were prepared from Malian adults who lived in a malaria endemic area. Antigen-specific antibody reactivity of the IgGs against the 1,827 proteins was evaluated using the AlphaScreen procedure. AlphaScreen is usually a protein-immobilization free procedure which is likely to maintain protein conformation better than the immobilization methods conventionally used in arrays and which allows high-throughput detection of protein-protein interactions15. Using the two data sets, the correlation between antibody responses and GIA activity was evaluated for each protein. We found that the antibody reactivity against three proteins was positively associated with GIA activities. Among the three, anti-C-terminal region of LSA3 (LSA3-C) responses showed the most significant correlation with the GIA activity. Human anti-LSA3-C-specific antibodies purified from Malian adult plasma also showed GIA activity. Previously, LSA3 has been evaluated as a pre-erythrocytic vaccine candidate, and exhibited a protective effect against sporozoite challenge in a chimpanzee16 and an monkey model17. Moreover, it has been shown that LSA3 genomic sequence is usually conserved in the isolates from diverse geographical areas compared to other blood-stage vaccine candidate18. In the present study, we have shown that LSA3 was expressed in blood-stage parasites judged by western blotting, and immunoelectron microscopy showed that LSA3 was localized to the dense granules of merozoites. OBSCN Taken together, we identified LSA3 as a novel blood-stage vaccine candidate through this study. Results Immunoreactivity of the recombinant proteins with Malian adult IgGs A total of 1 1,827 recombinant proteins mono-biotinylated at the N-terminus were expressed using WGCFS (Table S1). Antigen-specific IgG responses to these proteins were profiled by an AlphaScreen as described15 and shown schematically in Fig. S1. Immunoreactivity 1-Methyladenine of each test IgG against each antigen was decided, and a mean ASC (log-transformed AlphaScreen count) for each antigen was calculated..
