Controlled Human Malaria Infection by Mosquito Bites Using a Local Strain of Plasmodium Falciparum

Overview

The project aims to reproduce the malaria infection cycle using African mosquitoes, namely Anopheles gambiae and a local strain of Plasmodium parasites. The study will be conducted in two steps: (i) the production of infected mosquitoes and (ii) human infection with malaria parasites, known as Controlled Human Malaria Infection (CHMI). During the first step, Anopheles gambiae mosquitoes raised in the Centre de Recherches Médicales de Lambaréné (CERMEL) entomology laboratory in Lambaréné, Gabon, will be fed with blood collected from a person living in Lambaréné or the surrounding area who is positive for Plasmodium falciparum parasites, with the presence of asexual and sexual stages (responsible for malaria transmission) of the parasites in his/her blood. The Plasmodium-infected mosquitoes will be used two weeks later to infect healthy adult volunteers by mosquito bites under the supervision of the research team. After exposure, we will follow all exposed participants daily, starting on the fifth day after exposure. During the daily visits, we will perform malaria tests to detect the presence of malaria parasites in the blood of the participant. If participants test positive, they will receive usual antimalarial treatment if they simultaneously present malaria symptoms or if their parasitaemia is equal to or greater than 1,000 parasites per microlitre of blood. To ensure participants are effectively treated, we will perform two malaria tests after treatment, which should be negative. Otherwise, a different treatment will be administered. We will consider the process successful if we manage to infect (presence of malaria parasites in the blood) at least 50% of exposed participants. We also want the process to be safe, assuming that no unexpected clinical or biological events occur. Such an approach could contribute to a better assessment of the efficacy of antimalarial drugs and vaccine candidates, taking into account the specific characteristics of each endemic area.

Controlled Human Malaria Infection (CHMI) is a model to investigate immunity to malaria, diagnostics and, most importantly, antimalarial interventions. In the modern era, with the invention of parasite culture, it has almost exclusively used laboratory-adapted P. falciparum strains (NF54, 3D7, 7G8, NF135.C10, and NF166.C8) to infect volunteers either via injection of purified sporozoites or using infected mosquito bites ("flying syringes"). This model has greatly contributed to the evaluation of the efficacy of candidate antimalarial drugs or vaccines. However, one of its limitations lies in the fact that it does not take into consideration the genetic heterogeneity of Plasmodium infections typically presents in malaria-endemic regions. Indeed, P. falciparum strains in malaria-endemic regions are constantly under pressure from the host's human immune system and therapeutic interventions. This leads to genetically diverse strains of P. falciparum, different from the laboratory-adapted strains. Consequently, restricting CHMI to laboratory-adapted P. falciparum strains might not predict well the efficacy of vaccine candidates or antimalarial drugs in endemic settings, nor can it fully represent the biology of parasite field isolates and their potential interaction with hosts and other pathogens endemic in these areas. Therefore, it is postulated in this study that the CHMI model can be established in malaria-endemic regions using P. falciparum field isolates. This will not only contribute to the stringent evaluation of the efficacy of vaccine or antimalarial drug candidates in these settings but also to the understanding of the interaction between malaria and other diseases, particularly helminths. The main objective of the present work is thus to establish a safe protocol for the controlled infection of healthy adult human volunteers in an endemic area with local strains of P. falciparum via bites of infected A. gambiae s.s. Primarily, we will assess the infectivity to healthy adult Gabonese volunteers of A. gambiae Kisumu strain mosquitoes infected with local isolates of Pf. In addition, we will assess the safety of this CHMI procedure using locally P. falciparum-infected A. gambiae mosquitoes The project will be composed of two steps: the production of mosquitoes infected with P. falciparum (Clinical Trial Material) at the entomology laboratory of CERMEL following established SOPs, and CHMI by bites of Pf-infected mosquitoes on healthy volunteers. Pf-infected mosquitoes (abbreviated as Pf-iAgK mosquitoes) will be produced by infecting through membrane-feeding laboratory-raised A. gambiae Kisumu strain mosquitoes with a Pf gametocyte isolate obtained from a suitable donor. Consequently, only the successfully Pf-infected eligible batches of A. gambiae will be further used in the project. The CTM release criteria are: * negative PCR for Wuchereria bancrofti on the Pf gametocyte donor blood * negative PCR for O'nyong-nyong virus (ONNV) in the Pf gametocyte donor blood * full in vitro sensitivity of the Pf gametocyte donor isolates to the following antimalarial drugs: artesunate and lumefantrine * in vivo sensitivity by day 14 (+/- 1 day) * batch of mosquitoes with at least 40% infected mosquitoes (assessed by oocyst and sporozoite count) and a mean of at least 1000 sporozoites per mosquito The study will include twenty or thirty participants. First, ten healthy adult volunteers will be recruited to receive 5 Pf-infected mosquito bites each under direct supervision on day 0 (Cohort A1). If at least 50% of volunteers in Cohort A are found to be Pf-infected during the follow-up period, 10 more healthy adult volunteers will be enrolled to receive bites by the same number of Pf-infected A. gambiae mosquitoes using the same follow-up design as above (Cohort A2) but using a Pf isolate from one or more new donors. Data from Cohorts A1 and A2 will be combined to calculate infectivity rates. If less than 50% of volunteers in Cohort A1 are found to be Pf-infected during the follow-up, 10 more healthy adult volunteers will be enrolled to be bitten by double the number of Pf-infected A. gambiae mosquitoes (i.e., 10 mosquitoes) (Cohort B1) using Pf isolates from one or more new donors. If at least 50% of volunteers in Cohort B1 are found to be Pf-infected during the follow-up period, 10 more healthy adult volunteers will be enrolled to be bitten by the same number of Pf-infected A. gambiae mosquitoes (i.e., 10) (Cohort B2) but using Pf isolates from one or more new donors. Data from Cohorts B1 and B2 will then be combined to calculate infectivity rates. Further dose escalation (number of Pf-infected mosquito bites) may be considered via a potential amendment, depending on the safety and infectivity results in Cohort B1. In the study procedure, the sporozoites of Pf isolates will be inoculated into recipients via bites of PfiAgK mosquitoes. Recipients will be exposed to bites of infected mosquitoes for 10 minutes. After exposure, the number of fed and unfed mosquitoes will be recorded. Fed mosquitoes will be dissected to confirm the presence of sporozoites in the salivary glands. Any fed mosquitoes without sporozoites in the salivary glands, along with unfed mosquitoes, will be replaced by an identical number of infected mosquitoes for another round of 10 minutes of exposure until the required number of infected mosquitoes with sporozoites successfully take blood from the recipient. Each participant will be followed up for a period of 28 days, starting from day 5 post-exposure and qPCR (RT-qPCR based on 18S rRNA genes) and thick blood smear (TBS) will be performed to detect asexual blood stage parasitaemia. qPCR and TBS sampling will be performed daily until one of the malaria treatment criteria is met by day 28. Venous blood samples for immunology will be taken on days 0, 5, 11, 14, 28, and as applicable on the day of treatment before treatment administration. Venous blood samples will be taken for genetic and transcriptomic analyses on days 11, 14 and 28 or on the day of treatment before treatment administration.