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

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 International Center for Excellence in Research (ICER).

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 collaborations with external partners.

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.

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

Ethics Committee.

372 Towards Malaria Elimination - A Leap Forward

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)

vaccine that does not directly benefit a recipient, but instead benefits a community only when the proportion of the population that receives the vaccine achieves the threshold needed to reduce malaria prevalence and incidence. This threshold may vary between communities depending on the baseline intensity and ecology of malaria transmission. A TBV can be considered a "vaccine of solidarity," whereby the individual accepts vaccination to contribute to protection of his/her community and thereby ultimately to his/her own benefit. Before any decision to participate, individuals must balance the risks of immunization and the benefits for the community and ultimately themselves. To address this issue during clinical trials, candidate study participants must pass a test of comprehension that indicates the participant's understanding that the TBV has no potential to directly prevent their own infections. At present, there are few data in the literature on individual and community perceptions and understanding of TBV. Education on the mechanisms of action and potential community/

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Additionally, this community-based approach and vaccine coverage goal sets a high bar to achieve, even for a malaria vaccine. The vaccine needs to be efficacious not only in children but also in the majority of individuals in the community that contribute to transmission. The impact of factors such as malaria exposure, immunodeficiency, coinfection, pregnancy, and age on antibody responses will require careful study after qualification of promising TBV candidates. Thus, further research is needed to understand transmission dynamics within a community, durability and boosting of TBVs, and immunogenicity throughout all individuals

Furthermore, the DSF assay that experimentally exposes humans to mosquito bites raises ethical concerns and logistical burdens that may impact scientific objectives and study designs, and in some communities, recruitment and retention of volunteers. Mosquitoes used for DSF are reared in controlled and contained conditions to ensure the safety of the volunteers. While DSF has approval by ethical review committees, community perceptions should be considered to reassure participants and promote participant engagement in the process and rationale to prevent loss to follow-up that can compromise trial objectives. Our experience in Mali shows that strong community relationships and individual education in study design and goals have resulted in high participation and retention rates for studies that incorporate DSF assays.

The leading TBV candidates are either not exposed (Pfs25) to the immune system naturally or induce only modest responses naturally, and thus natural boosting may do little or nothing to extend antibody responses. Human antibodies should act quickly before degradation in the mosquito and before parasite traversal of midgut epithelium (~24 h) that limits its accessibility to antibody. The need for TBV to maintain high levels of potent antibody that preclude mosquito infection is the major hurdle for developing TBV. Today, several strategies are being pursued to overcome this hurdle, including the improved adjuvants and delivery systems discussed above. Additional antigens are also under exploration such as Pfs48/45 [54] and

As there is no existing human-based intervention that uniquely aims at blocking parasite transmission, such a tool could shift interest toward the interruption of transmission in comparison to the urgent need for interventions that protect and treat children under five and pregnant women. Once TBV tolerability and impact in the field are confirmed in advanced

individual benefits of TBV will be required before implementation.

PfHAP2 [55] that could expand the portfolio of TBV in future.

within a community.

Figure 4. General profile of a malaria transmission-blocking vaccine trial in Mali. This is general study process (flowchart) followed at the Malaria Research and Training Center (MRTC), Mali in carrying out a malaria vaccine study.

For community engagement, MRTC has built participative, durable, and confidence-based partnerships with communities at and around the study sites. Initially, community leaders comprising village heads, elderly, family heads, women's and youth's representatives, local health providers, and school teachers are consulted for permission to build research facilities and conduct clinical trials in the communities [53] (Figure 4). Subsequently, individual consents are obtained from interested volunteers according to international guidelines.

Local residents are fully involved and meaningfully impacted by the research activities. The presence of research teams and facilities, along with successful execution of clinical trials, has contributed to significant improvement of the local healthcare system and even the economy of the host villages. This positive impact has reinforced community confidence towards clinical trials and subsequently strengthened community engagement, positioning MRTC to successfully conduct TBV field clinical trials in Mali.

## 10. Bottlenecks and perspectives for TBVs

The future development and potential implementation of current TBV candidates will need to address important regulatory issues. A major issue involves the conceptual framework for a vaccine that does not directly benefit a recipient, but instead benefits a community only when the proportion of the population that receives the vaccine achieves the threshold needed to reduce malaria prevalence and incidence. This threshold may vary between communities depending on the baseline intensity and ecology of malaria transmission. A TBV can be considered a "vaccine of solidarity," whereby the individual accepts vaccination to contribute to protection of his/her community and thereby ultimately to his/her own benefit. Before any decision to participate, individuals must balance the risks of immunization and the benefits for the community and ultimately themselves. To address this issue during clinical trials, candidate study participants must pass a test of comprehension that indicates the participant's understanding that the TBV has no potential to directly prevent their own infections. At present, there are few data in the literature on individual and community perceptions and understanding of TBV. Education on the mechanisms of action and potential community/ individual benefits of TBV will be required before implementation.

Additionally, this community-based approach and vaccine coverage goal sets a high bar to achieve, even for a malaria vaccine. The vaccine needs to be efficacious not only in children but also in the majority of individuals in the community that contribute to transmission. The impact of factors such as malaria exposure, immunodeficiency, coinfection, pregnancy, and age on antibody responses will require careful study after qualification of promising TBV candidates. Thus, further research is needed to understand transmission dynamics within a community, durability and boosting of TBVs, and immunogenicity throughout all individuals within a community.

Furthermore, the DSF assay that experimentally exposes humans to mosquito bites raises ethical concerns and logistical burdens that may impact scientific objectives and study designs, and in some communities, recruitment and retention of volunteers. Mosquitoes used for DSF are reared in controlled and contained conditions to ensure the safety of the volunteers. While DSF has approval by ethical review committees, community perceptions should be considered to reassure participants and promote participant engagement in the process and rationale to prevent loss to follow-up that can compromise trial objectives. Our experience in Mali shows that strong community relationships and individual education in study design and goals have resulted in high participation and retention rates for studies that incorporate DSF assays.

For community engagement, MRTC has built participative, durable, and confidence-based partnerships with communities at and around the study sites. Initially, community leaders comprising village heads, elderly, family heads, women's and youth's representatives, local health providers, and school teachers are consulted for permission to build research facilities and conduct clinical trials in the communities [53] (Figure 4). Subsequently, individual con-

Figure 4. General profile of a malaria transmission-blocking vaccine trial in Mali. This is general study process (flowchart) followed at the Malaria Research and Training Center (MRTC), Mali in carrying out a malaria vaccine study.

Local residents are fully involved and meaningfully impacted by the research activities. The presence of research teams and facilities, along with successful execution of clinical trials, has contributed to significant improvement of the local healthcare system and even the economy of the host villages. This positive impact has reinforced community confidence towards clinical trials and subsequently strengthened community engagement, positioning MRTC to success-

The future development and potential implementation of current TBV candidates will need to address important regulatory issues. A major issue involves the conceptual framework for a

sents are obtained from interested volunteers according to international guidelines.

fully conduct TBV field clinical trials in Mali.

374 Towards Malaria Elimination - A Leap Forward

10. Bottlenecks and perspectives for TBVs

The leading TBV candidates are either not exposed (Pfs25) to the immune system naturally or induce only modest responses naturally, and thus natural boosting may do little or nothing to extend antibody responses. Human antibodies should act quickly before degradation in the mosquito and before parasite traversal of midgut epithelium (~24 h) that limits its accessibility to antibody. The need for TBV to maintain high levels of potent antibody that preclude mosquito infection is the major hurdle for developing TBV. Today, several strategies are being pursued to overcome this hurdle, including the improved adjuvants and delivery systems discussed above. Additional antigens are also under exploration such as Pfs48/45 [54] and PfHAP2 [55] that could expand the portfolio of TBV in future.

As there is no existing human-based intervention that uniquely aims at blocking parasite transmission, such a tool could shift interest toward the interruption of transmission in comparison to the urgent need for interventions that protect and treat children under five and pregnant women. Once TBV tolerability and impact in the field are confirmed in advanced clinical trials, consideration must be given for how this vaccine should be implemented alongside other malaria control tools. Ultimately, TBVs are envisioned as one tool in the toolbox of interventions, including antimalarial drugs, vector control measures, and other vaccines, that will be required for malaria elimination.

Acronyms

AnaPN1 Anopheles gambiae aminopeptidase 1

CHMI Controlled human malaria infection

DMSFA Standard membrane-feeding assay

ELISA Enzyme-linked immunosorbent assay EPA Pseudomonas aeruginosa exoprotein A.

EPI Expanded Programme on Immunization

ICER International Center for Excellence in Research

LMIV Laboratory of Malaria Immunology and Vaccinology


ICH International Conference on Harmonization

DSF Direct skin-feeding assay

GCP Good Clinical Practices

GSK GlaxoSmithKline

GPI Glycosylphosphatidylinositol

IMX313 C4 bp oligomerization domain

IRB Institutional Review Board IRS Indoor residual spraying ITN Insecticide-treated nets

LikM Modified lichenase carrier

MPL 3-O-desacyl-4<sup>0</sup>

MalERA Malaria Eradication Research Agenda

Flp Flippase

AS01 GSK's adjuvant system containing liposome, 3-O-desacyl-4<sup>0</sup>

CELTOS Cell-traversal protein for ookinetes and sporozoites

CRISPR-associated protein 9 (Cas9)

FMPOS Faculty of Medicine, Pharmacy and Odonto-Stomatology

lipid A (MPL) and Quillaja saponaria Molina saponin fraction 21 (QS21)

Malaria Transmission-Blocking Vaccines: Present Status and Future Perspectives

CRISPR/Cas9 Clustered regularly interspaced short palindromic repeats (CRISPR) and


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