Studies on asexual stage immunity to P. falciparum 1) Evaluate the merozoite antigen PfRH5 as a vaccine candidate. Our collaborators at Oxford University (Dr. Simon Draper et al.) are pursuing this protein as a vaccine candidate and we have contributed to various pre-clinical studies using our standardized growth-inhibition assay (GIA). A clinical trial using recombinant PfRH5 protein showed about 20% reduction in parasite growth. This is the first time that positive results have been seen with a blood stage vaccine and we have completed GIA studies from this trial. 2) Quantitation of the human responses to this vaccine combined with the quantitative GIA results have provided a marker which needs to be achieved to see greater protection in humans. The increment is approximately 5-fold and efforts to reach this are under development. 3)Part of this effort has shown that there are different epitopes on the PfRH5 surface and that some are critically important for function of the protein. Characterizing monoclonal antibodies (mabs) to this protein has allowed identification of mabs which themselves do not show GIA but do enhance the activity of other mabs. The functionality of these non-neutralizing antibodies seems to be to slow the invasion of merozoites into red cells. 4) In addition, we have initiated a detailed study of immune responses in field sites to PfRH5 as well as other associated molecules which are part of an invasion complex with this molecule - PfCyRPA, PfRipr, etc. Antibody titers in Malians to these proteins are very low but we are isolating their specific antibodies and are studying their functionality. 5) We have collaborated with Oxford investigators on passive transfer of monoclonal antibodies to PfRH5 into Aotus monkeys then challenged with malaria parasites. We have established a GIA threshold required for protection in vivo in this NHP system. 6) PfRH5 vaccines are now being tested in Tanzania and we are conducting GIA analysis of the human responses. 7) Other targets of merozoite immunity - AMA1. We have collaborated with Dr. Lou Miller to show that using AMA1 in conjunction with its partner protein RON2 produces antibodies with greater activity in the GIA assay. 8) We are also collaborating with Oxford University (Dr. Adrian Hill) on a major EU-funded project to develop a 3-stage multi-component vaccine for P. falciparum. 9) We have started a collaboration with Drs. Peter Crompton/Joshua Tan, and Robert Seder to do high throughput identification of human monoclonal antibodies to whole parasites and recombinant proteins of malaria using B cells from Malians as the source of the antibodies. 10) We are also collaborating with Dr. Richard Eastman (NCATS)on additional monoclonal antibody selection to PfRH5 and other parasite molecules using selected human and camelid libraries. 11) We have identified DNA aptamers which can specifically identify piperaquine and mefloquine, two of the partner drugs for artemisinin used for treatment of malaria around the world. These sensors now provide a platform for the development of a kit to detect these drugs in human blood and to test drug tablets for potency. 12) We have initiated studies of Plasmodium vivax to identify a biomarker of infection to improve detection kits. Studies on parasite sexual stages and transmission blocking vaccine candidates: 1)We have developed quantitative methodology for analysis of the standard mosquito membrane feeding assay (SMFA) to evaluate transmission blocking activity. The standard assay to evaluate the ability of antibodies to block transmission to mosquitoes is the SMFA, and we have performed an in depth study of this assay in order to have confidence in assessing potential transmission blocking vaccine (TBV) candidates. We have worked with the Biostatistics group at NIAID to develop a model of the assay and more recently we have shown for the first time that we can predict the impact of a specific antibody on the reduction of P. falciparum parasite prevalence in mosquitoes based on the reduction in oocysts in an SMFA and the number of oocysts in control mosquitoes. Reduction in malaria prevalence in mosquitoes by an antibody is key to reducing transmission in the field. 2) Search for and evaluate new possible transmission blocking vaccine candidates. Using SMFA we have evaluated a number of potential transmission blocking vaccine candidates. We have tested antibodies to significant numbers of sexual stage and mosquito vaccine candidates to compare their activity using quantitative measurements of antibody concentration and SMFA. 3) We have compared the number of oocysts with the number of sporozoites in the mosquito salivary gland. We have shown that there is a strong concordance of oocysts and salivary gland sporozoites in the SMFA. 4) Construction and evaluation of human/humanized monoclonal antibodies (mabs) to several sexual stage parasite proteins. We are currently comparing several mabs to Pfs25 and other candidates as a foundation for human clinical trials. Passive administration of these antibodies will allow evaluation of transmission blocking in vivo and might eventually be used in elimination campaigns. We are comparing them in SMFA, mapping their epitopes, and determining their binding properties. 5) We are continuing studies to examine the transcriptional pattern of P. falciparum gametocytes during differentiation in culture using RNASeq. We have obtained data on RNA transcription from gametocytes in culture and we are currently analyzing the data to identify markers which will correlate with the number of oocysts in the mosquito. In addition as part of this work we are seeking genetic targets which determine whether the gametocyte develops as a male or female. 6) We are continuing a series of experiments to determine what serum-derived factors are necessary for successful differentiation of gametocytes so that robust numbers of oocysts can be obtained after mosquito infection. Mass spectrometry of good and bad sera have shown consistent differences between them (Dr. M Llinas collaboration). 7) We have initiated studies on PfHAP2 as a novel transmission blocking vaccine candidate and we are starting new studies to identify other transmission blocking vaccine candidates with the group at Ehime University in Japan (Dr. Taka Tsuboi). 8) We are part of a consortium of investigators led by Oxford University (S. Biswas) and funded by the EU to develop a transmission blocking vaccine. Field studies of malaria transmission in Mali 1) We have assessed the presence of asexual and sexual stage parasites in residents of Kenieroba, Mali throughout the year. In the spring of 2013 we initiated a new study (NIH 13-I-N107) to address the limited information on transmission in malaria endemic areas, and in 2014 we completed the field aspect of this study.This work has been completed and published 2)We showed for the first time that increasing P.falciparum longitudinal prevalence throughout the year was associated with decreasing risk of clinical malaria. This suggests that those with persistent parasite carriage acquire stronger protective immunity. 3) We added a new barcoding procedure based on a methodology developed at Harvard University to determine how many different clones of parasites an individual was carrying. This allowed us to show that at least 70% of infections were polygenomic, so that most people in the population are carrying more than one parasite clone at any given time. 4) Using RNA collections from the same study, we used a qRT-PCR procedure to identify Pfs25 mRNA specific for gametocytes. More than 80% of people with parasites also have detectable gametocytes so that no one group can be uniquely targeted for interventions.
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