8. Measuring vaccine activity

Unlike other vaccines where controlled human malaria infection (CHMI) studies are useful for assessing efficacy, field assessment of TBV efficacy currently requires mosquito-feeding assays on individuals living in malaria-endemic areas. Several mosquito-feeding assays can be utilized to assess the capacity of vaccine-induced antibodies to interfere with mosquito infectivity including direct skin-feeding (DSF) assay, direct membrane-feeding assay (DMFA), and standard membrane-feeding assay (SMFA). The DMFA entails feeding of laboratory-reared uninfected mosquitoes on venous blood immediately after collection from study participants (Figure 2A). Feeding occurs through various types of membranes, such as pig intestine or Parafilm®, to access infected blood housed in a heated chamber that attracts mosquitoes. This method takes the diversity of infection in the population into account. The standard membrane-feeding assay (SMFA) is the gold-standard technique for functional evaluation of antibodies in TBV studies, given its use of a well-characterized laboratory parasite isolate and mosquito line that lend themselves to standardization. Mosquitoes feed on cultured gametocytes together with either volunteer serum or purified immunoglobulin (Figure 2B). SMFA is similar to DMFA in the machinery and process for feeding but fails to capture parasite diversity effects on vaccine activity.

7. Field studies of TBVs and Malian experiences

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transmission in the communities living in malaria-endemic areas.

quality of data according to established procedures.

8. Measuring vaccine activity

trials of malaria vaccines.

Field clinical trials are a major component of TBV development. TBV candidates are generally discovered in laboratories in the North with extensive infrastructure for modern biological sciences to conduct discovery research. After passing preclinical evaluations, TBV candidates must be tested in humans to qualify as viable vaccines. Safety and tolerability of the products are assessed first, generally in malaria-naïve individuals in non-endemic countries during a first-in-human phase 1 study. If the product meets acceptable safety and tolerability criteria, then it advances to Phases I, II, and III field clinical trials, which often means evaluation in malaria-endemic settings. Field studies are essential for assessing interruption of malaria

A field clinical trial is not just a simple study but rather a multifaceted activity that builds on strong partnerships between research institutions and affected communities. Various capabilities are required for successful clinical trials, including confidence-based collaborative research teams, facilities, equipment, written procedures, training programs, community engagement, collaboration with ethics review committees, and collaborations with health and political authorities. The partnerships include vaccine inventors, developers, sponsors/funders, and institutions that have appropriate capacities and experience in conducting field clinical

The main components of a TBV field clinical trial comprise immunization of study volunteers with prime and boost doses, intensive safety follow-up and reporting, mosquito-feeding assays, and the measurement of antibody responses using enzyme-linked immunosorbent assay (ELISA) for titers and standard membrane-feeding assay (SMFA) for activity. The immunization of volunteers is a major event that involves professionals with sundry expertise. The professionals include clinicians who assess volunteers for inclusion/exclusion criteria and monitor their health after receiving the vaccination, pharmacists who manage the randomization list as well as vaccine preparation, physicians who administer the vaccines, and intensivists who provide care for any post-immunization emergency. Medical biologists ensure proper biological sample collection, processing, transport, and storage as well as immediate measurement of biological parameters. Medical entomologists perform mosquito-feeding assays and associated dissections for endpoint analysis, and data managers enter and ensure

Unlike other vaccines where controlled human malaria infection (CHMI) studies are useful for assessing efficacy, field assessment of TBV efficacy currently requires mosquito-feeding assays on individuals living in malaria-endemic areas. Several mosquito-feeding assays can be utilized to assess the capacity of vaccine-induced antibodies to interfere with mosquito infectivity including direct skin-feeding (DSF) assay, direct membrane-feeding assay (DMFA), and standard membrane-feeding assay (SMFA). The DMFA entails feeding of laboratory-reared uninfected mosquitoes on venous blood immediately after collection from study participants In DSF assays, cups of field-adapted, laboratory-reared mosquitoes are fed on the skin of human volunteers to assess the ability of vaccine antibody responses to block malaria transmission in near-natural conditions (Figure 3). In a recent advance, the Malaria Research and Training Center (MRTC) in Bamako (Mali) and the Laboratory of Malaria Immunology and Vaccinology (LMIV) at NIAID/NIH, in Rockville, MD (USA) have established the infrastructure, logistics, and safety database to support scale up of DSF assays on a community-wide basis (Figure 3). These DSF assays use a line of locally caught Anopheles coluzzii recently adapted for breeding in a contained insectary. In these studies [52], uninfected mosquitoes (generally 30–60 per assay) are fed on study volunteers on a regular basis during the malaria transmission season, with the expectation that an effective vaccine will reduce the number of infected mosquitoes compared to controls. We believe DSFs, in which insectary-raised clean mosquitoes are directly fed on infected individuals, may be more likely to be predictive of an intervention's impact on transmission than membrane feeds.

Figure 2. Membrane-feeding assays. (A) Overall setup of a direct membrane-feeding assay (DMFA). DMFA was performed at the Malaria Research and Training Center (MRTC), Mali with mosquitoes feeding through a membrane on whole venous blood taken in citrate-phosphate-dextrose or heparin from infected donors. The mosquitoes used were F1 or F2 progeny of wild-caught mosquitoes, or MRTC colony-bred mosquitoes (Anopheles coluzzii) maintained for many generations after local capture. The gametocyte source was fresh venous blood collected from infected study volunteers in Mali. (B) Standard membrane-feeding assay (SMFA) showing an individual feeding chamber. SMFA performed at the Laboratory of Malaria Immunology and Vaccinology (LMIV), National Institute of Allergy and Infectious Diseases (NIAID)/National Institutes of Health (NIH), Rockville, MD, USA with mosquitoes feeding through a membrane on laboratory-cultured parasites (gametocytes) suspended in media with immune or nonimmune serum/plasma or IgG. The mosquitoes used were an established laboratory strain (commonly Anopheles stephensi).

activity, which are respectively the ratio of the proportions of infected mosquitoes and the

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The measurement of antibody titers during field clinical trials of TBVs (generally by ELISA) is required to assess the immunogenicity of vaccine candidates. Samples are assayed to detect any pre-existing immunity against vaccine candidates, to determine vaccine immunogenicity after prime and boost doses, and to monitor the decay of antibody titers during the follow-up. In addition, antibody titer data can be linked with mosquito-feeding assay data to determine the correlation between antibody production and functionality. Many further investigations can be performed with samples collected from the study volunteers for other exploratory objectives.

The achievement of all field activities requires an institution endowed with strong capacities and trained staff. Malaria Research and Training Center (MRTC), founded in 1992 at the Department of Parasitic Diseases Epidemiology based at the University of Sciences, Techniques and Technologies of Bamako (USTTB) in Mali, is a leading institution in conducting field clinical trials of TBVs in Africa. The center is the result of a partnership between the Malian government and US National Institutes of Health (NIH) to build capacity in malaria research and training in Mali. The USTTB collaboration has been designated by NIH as an

MRTC comprises six equipped and autonomous clinical trial sites located in malaria-endemic Malian villages that fulfill International Conference on Harmonization (ICH) requirements and adhere to Good Clinical Practices (GCP) and a central laboratory in Bamako (the capital city of Mali) where several teams are based. The MRTC infrastructure includes a clinical laboratory certified by the College of American Pathologists and an equipped insectary, among others. In addition, the center has 15 other field sites that host epidemiological studies and other collab-

From 2003 to 2016, MRTC completed 13 asexual stage vaccine clinical trials and one TBV field clinical trial evaluating Pfs25H-EPA/Alhydrogel® (Bancoumana, Mali, from 2013 to 2016, NCT01867463) in collaboration with LMIV at NIH. Field clinical trials of six more candidate TBVs are ongoing: Pfs25M-EPA/Alhydrogel® and Pfs230D1M-EPA/Alhydrogel®, as well as the combination of Pfs25M-EPA/Alhydrogel® + Pfs230D1M-EPA/Alhydrogel® (Bethesda, USA and Bancoumana, Mali; started in 2015, NCT02334462), and Pfs25M-EPA/AS01, Pfs230D1M-EPA/AS01, and the combination of Pfs25M-EPA/AS01 + Pfs230D1M-EPA/AS01 (Bamako, Doneguebougou and Bancoumana, Mali; started in 2016, NCT02942277). The success of these studies depends on MRTC's technical capacities and strength in community engagement. Dynamic MRTC teams execute intense programs of volunteer immunization and thorough follow-up (Figure 4), as well as high-throughput processing of samples in laboratories, rearing of mosquitoes, mosquito-feeding assays, dissection of mosquitoes, real-time data entry and cleaning, and constant transportation of persons, samples, and materials between central laboratory and study sites.

9. The Malaria Research and Training Center (MRTC) field trials

mean counts of oocysts, between test and control mosquitoes.

International Center for Excellence in Research (ICER).

orations with external partners.

experience

Figure 3. Direct skin feeds (DSF). DSF was performed at the Malaria Research and Training Center (MRTC), Mali at the study sites. MRTC colony-bred mosquitoes (Anopheles coluzzii) raised in containment fed directly on skin of consenting study participants. The gametocyte source was infected study volunteers in Mali. Ethical approvals were obtained from both the NIAID/NIH IRB (National Institute of Allergy and Infectious Diseases/National Institutes of Health, Rockville, MD, USA Institutional Review Board) and the Mali FMPOS (Faculty of Medicine, Pharmacy and Odonto-Stomatology) Ethics Committee.

For all these methods (SMFA, DMFA, DSF), mosquitoes are dissected about a week after feeding for oocyst detection and counting by microscopy. The results of the mosquito-feeding assay allow the calculation of transmission-blocking activity and transmission reducing activity, which are respectively the ratio of the proportions of infected mosquitoes and the mean counts of oocysts, between test and control mosquitoes.

The measurement of antibody titers during field clinical trials of TBVs (generally by ELISA) is required to assess the immunogenicity of vaccine candidates. Samples are assayed to detect any pre-existing immunity against vaccine candidates, to determine vaccine immunogenicity after prime and boost doses, and to monitor the decay of antibody titers during the follow-up. In addition, antibody titer data can be linked with mosquito-feeding assay data to determine the correlation between antibody production and functionality. Many further investigations can be performed with samples collected from the study volunteers for other exploratory objectives.
