title: Robust, reproducible, industrialized, standard membrane feeding assay for assessing the transmission blocking activity of vaccines and drugs against Plasmodium falciparum.
abstract: BACKGROUND
  A vaccine that interrupts malaria transmission (VIMT) would be a valuable tool for malaria control and elimination. One VIMT approach is to identify sexual erythrocytic and mosquito stage antigens of the malaria parasite that induce immune responses targeted at disrupting parasite development in the mosquito. The standard Plasmodium falciparum membrane-feeding assay (SMFA) is used to assess transmission-blocking activity (TBA) of antibodies against candidate immunogens and of drugs targeting the mosquito stages. To develop its P. falciparum sporozoite (SPZ) products, Sanaria has industrialized the production of P. falciparum-infected Anopheles stephensi mosquitoes, incorporating quantitative analyses of oocyst and P. falciparum SPZ infections as part of the manufacturing process.
  
  
  METHODS
  These capabilities were exploited to develop a robust, reliable, consistent SMFA that was used to assess 188 serum samples from animals immunized with the candidate vaccine immunogen, Pfs25, targeting P. falciparum mosquito stages. Seventy-four independent SMFAs were performed. Infection intensity (number of oocysts/mosquito) and infection prevalence (percentage of mosquitoes infected with oocysts) were compared between mosquitoes fed cultured gametocytes plus normal human O(+) serum (negative control), anti-Pfs25 polyclonal antisera (MRA39 or MRA38, at a final dilution in the blood meal of 1:54 as positive control), and test sera from animals immunized with Pfs25 (at a final dilution in the blood meal of 1:9).
  
  
  RESULTS
  SMFA negative controls consistently yielded high infection intensity (mean = 46.1 oocysts/midgut, range of positives 3.7-135.6) and infection prevalence (mean = 94.2%, range 71.4-100.0) and in positive controls, infection intensity was reduced by 81.6% (anti-Pfs25 MRA39) and 97.0% (anti-Pfs25 MRA38), and infection prevalence was reduced by 12.9 and 63.5%, respectively. A range of TBAs was detected among the 188 test samples assayed in duplicate. Consistent administration of infectious gametocytes to mosquitoes within and between assays was achieved, and the TBA of anti-Pfs25 control antibodies was highly reproducible.
  
  
  CONCLUSIONS
  These results demonstrate a robust capacity to perform the SMFA in a medium-to-high throughput format, suitable for assessing large numbers of experimental samples of candidate antibodies or drugs.
authors.affiliation: Sanaria Inc., 9800 Medical Center Drive, Rockville, MD, 20850, USA. tli@sanaria.com.
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.affiliation: Sanaria Inc., 9800 Medical Center Drive, Rockville, MD, 20850, USA. aeappen@sanaria.com.
authors.forename: Abraham G
authors.initials: AG
authors.lastname: Eappen
authors.affiliation: Sanaria Inc., 9800 Medical Center Drive, Rockville, MD, 20850, USA. arichman@sanaria.com.
authors.forename: Adam M
authors.initials: AM
authors.lastname: Richman
authors.affiliation: Sanaria Inc., 9800 Medical Center Drive, Rockville, MD, 20850, USA. pbillingsley@sanaria.com.
authors.forename: Peter F
authors.initials: PF
authors.lastname: Billingsley
authors.affiliation: Sanaria Inc., 9800 Medical Center Drive, Rockville, MD, 20850, USA. yabebe@sanaria.com.
authors.forename: Yonas
authors.initials: Y
authors.lastname: Abebe
authors.affiliation: Protein Potential LLC, 9800 Medical Center Drive, Rockville, MD, 20850, USA. mli@sanaria.com.
authors.forename: Minglin
authors.initials: M
authors.lastname: Li
authors.affiliation: Sanaria Inc., 9800 Medical Center Drive, Rockville, MD, 20850, USA. dpadilla@sanaria.com.
authors.forename: Debbie
authors.initials: D
authors.lastname: Padilla
authors.affiliation: Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Suite E6003, Baltimore, MD, 21205, USA. monedita@gmail.com.
authors.forename: Isabel
authors.initials: I
authors.lastname: Rodriguez-Barraquer
authors.affiliation: Sanaria Inc., 9800 Medical Center Drive, Rockville, MD, 20850, USA. ksim@protpot.com.|Protein Potential LLC, 9800 Medical Center Drive, Rockville, MD, 20850, USA. ksim@protpot.com.
authors.forename: B Kim Lee
authors.initials: BK
authors.lastname: Sim
authors.affiliation: Sanaria Inc., 9800 Medical Center Drive, Rockville, MD, 20850, USA. slhoffman@sanaria.com.
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Hoffman
mesh_terms: D000818:Animals; D000852:Anopheles / Q000502:physiology*; D000962:Antimalarials / Q000494:pharmacology*; D001681:Biological Assay / Q000379:methods*; D005247:Feeding Behavior; D005260:Female; D017780:Malaria Vaccines / Q000494:pharmacology*; D008566:Membranes / Q000502:physiology; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000276:immunology*
chemical_list: D000962:Antimalarials; D017780:Malaria Vaccines