ASTMH 2020 Annual Meeting (virtual): Day 5
MESA Correspondents bring you cutting-edge coverage from the virtual ASTMH 2020 Annual Meeting
Day 5: Thursday, 19th November 2020
Symposium #117: Vaccines Against Placental Malaria
The organizers, Stephanie Yanow and Nicaise Tuikue Ndam, introduced this discussion topic by emphasizing the importance of developing a vaccine against placental malaria. Pregnant women are especially susceptible to malaria which affects infant birth weight alongside maternal anemia and can lead to abortions. The unique sequestration of Plasmodium-infected erythrocytes to the placenta poses detrimental risks to the mother and baby and therefore vaccines suitable for pregnant women are necessary.
Arnaud Chȇne (French Institute of Health and Medical Research, Paris, France) updated the audience on progress in the development of a placental malaria vaccine. About 30 million women become pregnant in malaria-endemic areas annually, and this malaria-susceptible group requires some form of protection. He further noted that primigravidas have little immunity to placental malaria while multigravidas do. Previous reports demonstrated that infected erythrocytes express the VAR2CSA variant of P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) which allows them to bind to chondroitin sulfate A (CSA) during parasite sequestration in the placenta and that this process is essential to the pathogenesis of maternal malaria. A VAR2CSA-based vaccine could therefore offer potential protection to improve pregnancy outcomes, the main goal of the PRIMVAC project. Different vaccines were formulated using different constructs, expression systems, and adjuvants. Immunogenicity was assessed, and elicited antibodies were evaluated for their specificity and affinity to VAR2CSA cross-reactivity, and cross-inhibitory capacity against heterologous strains. The most suitable candidate spanned the Duffy binding-like domains 1 and 2 (DBL1-2) from 3D7 expressed in E. coli SHuffle (PRIMVAC). Phase 1 clinical trials on women aged 18-35 were conducted showing that PRIMVAC did not elicit serious adverse reactions, was highly immunogenic and induced good reactivity and inhibitory activity against the homologous VAR2CSA-expressing strain. In contrast, it had only weak cross-reactivity and no cross-inhibition against heterologous strains.
Nicaise Ndam (University of Ghana, Ghana) gave an overview of the "Malaria Vaccine Technology Roadmap" from 2015 to 2030 and prospects for vaccines against P. falciparum and P. vivax. These vaccines should have more than 70% efficacy against clinical malaria, reduce parasite transmission and significantly decrease the incidence of infection to achieve elimination in several areas. The vaccine candidate PAMVAC is based on the minimal CSA binding domain (DI1-ID2a) of VAR2CSA from FCR3. PAMVAC was evaluated in Phase I clinical trial with two cohorts, one in Germany and another in Benin. Antibodies from vaccines recognized the homologous parasite FCR3 but had limited cross-reactivity to heterologous VAR2CSA strains. Ndam discussed the limitations and obstacles for further clinical development of the vaccine, particularly the challenges posed by VAR2CSA polymorphisms, the need for inducing long-lasting immunity and the logistical challenges of measuring vaccine efficacy in pregnant women.
Following these highlights, Justin Doritchamou (National Institute of Allergy and Infectious Disease, USA) in his presentation “Aotus model is superior to rodent models in predicting human immune response to VAR2CSA vaccine” described a different animal model to measure the efficacy of VAR2CSA-based vaccines. Aotus is biologically closer to humans compared to mice and rabbits and is, therefore, a suitable model for vaccine studies. IgG elicited to the PAMVAC and PRIMVAC vaccine candidates was studied in the Aotus model with similar results to those observed in the human trials. The antibodies had good functional activity against homologous parasites but little cross-inhibition to heterologous parasites. The study also showed that malaria infection in pregnant vaccinated animals failed to boost functional antibodies. Based on these findings, Aotus is a preferred model compared to rodents and may provide a more accurate prediction of vaccine efficacy in humans.
Finally, Stephanie Yanow (University of Alberta, Canada) discussed a cross-species vaccine approach to elicit VAR2CSA antibodies. Analyses from her lab showed that, unlike in African women, antibodies to VAR2CSA are not parity-dependent in Colombian women. Serum antibodies from men and children could recognize VAR2CSA. In addition, a blocking parasite adhesion effect to in vitro CSA was noted with antibodies from Colombian men and children. These two last observations raised the question of how VAR2CSA antibodies are acquired outside of pregnancy and suggested immunogenicity and potential cross-reactivity with P. vivax proteins. She presented data mapping a conformational epitope in the P. vivax PvDBP protein that mediates cross-reactivity to VAR2CSA. This epitope was recapitulated using a conformationally constrained peptide. When conjugated to a carrier protein, this peptide elicited cross-reactive antibodies to VAR2CSA in mice and rabbits, and shows promise as a vaccine candidate against placental malaria.
Symposium #119: Cross-Disciplinary Sciences to Understand Malaria Vaccine Immunity
Carlota Dobaño (ISGlobal, Spain) in her talk “RTS,S vaccine immunity and immune system development in African children” presented findings from a phase III multi-centre trial with around 2000 children (age 6-12 weeks and 5-17 month) in seven countries. The findings showed that RTS,S/AS01E-induced protection against malaria involved IgG against the C-terminal region besides the NANP repeat region of the circumsporozoite protein (CSP). CSP-specific Th1 cytokine signatures were associated with RTS,S/AS01E-induced protection against malaria, while Th2 signatures correlated with risk. Importantly, immunogenicity was shown to be lower in infants than children. Another finding was that the baseline cellular activation status was associated with RTS,S/AS01E antibody response. Lastly, interferon transcriptional signatures were associated with vaccine-induced protection.
Stephen Hoffman (Sanaria Inc., United States) in his talk on “Progress towards understanding and harnessing the complexity of protective sporozoite vaccine immunity against malaria” called for a new cross-disciplinary approach and advancement in immunological insights. Although phenomenal progress has been made, the understanding of the immunological mechanisms of protection induced by whole PfSPZ vaccines has changed little in the past two decades’ due to being mostly based on animal studies. Hoffman presented results from vaccine studies in terms of efficacy, protection, and duration. Four studies conducted in Mali and Burkina Faso showed achievement of 50% efficacy and sustained efficacy for 18 months in Burkina Faso. In 2021, a phase III trial with the PfSPZ vaccine will start aiming for licensure and marketing authorization. Although not yet achieving 100% efficacy, a foundation would be laid out for further improvements. To move forward, it would be key to understand induction and effector phases of protective immunity and to establish biomarkers of protection, being able to interrogate the immune responses in liver, spleen and essential lymph nodes. To do so, Hoffman proposes a different approach with, firstly, studies on P. knowlesi and P. falciparum in rhesus macaques and Aotus species to establish mechanisms and biomarkers of induction and protection t, and then clinical trials in humans to further corroborate the findings.
In the presentation “Do differences make a difference: using systems immunology to dissect natural and engineered adjuvants”, John Tsang (NIAID/NIH, United States) addressed questions on predictability of immune response outcomes, type of predicted parameters and shared mechanism across different types of immune responses. The presented data suggest the existence of a baseline predictor that can be shared across different vaccines. Evidence on yellow fever indicated that the baseline predictor would not include pre-existing immunity. To further investigate this, they applied cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq). The results suggested a two-stage immune response model with high and low responders. Applying CITE-seq and other methods provided a better understanding of baseline predictive signatures and allowed the discovery of natural adjuvants. Moreover, the application of multimodal single cell analyses allowed for easier interpretation of immunological cell clusters, for virtual sorting and denoising, as well as linking clinic to cell surfaces and transcriptomic phenotypes.
Purvesh Khatri (Stanford University, United States) shared in his presentation “Leveraging biological, clinical and technical heterogeneity in public data to accelerate translational medicine” a new framework for predicting clinical outcomes prior to treatment or vaccination in heterogeneous populations. He described the traditional approach that strives for reduced heterogeneity as a ‘fundamental flaw’ of the current paradigm in biomedical and translational research. There is no technical variation and most research does not capture heterogeneity of the disease and therefore results produced cannot be generalized. Khatri's proposed solution to this phenomenon is to embrace heterogeneity by using different publicly available datasets, different treatments, and different technologies, so results can be generalized. He has developed a model that leverages heterogeneity of biological data available online. The model was applied and validated in several use cases, leading to new discoveries that currently move toward commercialization. In the end, Khatri emphasized the need for sharing data in order to unravel biological, clinical and technical heterogeneity and to provide the opportunity to identify new signatures/biomarkers and develop new interventions.
Symposium #121: Comprehensive Surveillance in the Setting of a Dramatic Decline in Malaria Following Sustained Control Interventions in a Historically High Transmission Area of Uganda: From Mosquito to Human and Back Again
Alex K. Musiime (Infectious Diseases Research Collaboration, Uganda) presented work on the changes in malaria vector biology and bionomics following intensified vector control in Tororo, Uganda, an area with a high malaria transmission rate. In the study, malaria transmission indoors and outdoors, before and after intensified vector control were analysed along with vector behaviour. Two cohort studies i.e. Program for resistance, immunology, surveillance, and modelling of malaria 1 (PRISM 1) (2011-2017) and PRISM 2 (2017-2019) were carried out with all participants given long-lasting insecticidal nets (LLINs) at the start of the study. Results showed that indoor residual spraying intervention resulted in a sustained decrease in human-biting and infective rates. However, the intervention resulted in a shift to early evening biting parallel with an increase in outdoor biting. The vector control had a negative effect on numbers An. gambiae s.s and An. funestus but resulted in an increase of Anopheles species. Although successful in decreasing vector abundance, the study also highlighted the need to incorporate tools that reduce outdoor biting in the fight against malaria transmission.
Joaniter I. Nankabirwa (Makerere University, Uganda) presented work on malaria transmission, infection and disease following sustained indoor residual spraying of insecticide in Tororo, Uganda. To curb the high transmission rate in this district vector control measures were scaled up, long-lasting insecticide nets (LLIN) in 2013and indoor residual spraying (IRS) in 2014. The study set out to evaluate the impact of intensive vector control on various clinical metrics in two serial cohort studies. Results showed that in children between 0.5 to 10 years, IRS intervention led to a decrease in monthly malaria incidence from 2.66 to 0.05 after 5 years. Also, there was a decline in the prevalence of anaemia, from 34.6% to 5.1%, in both microscopic and submicroscopic infection. However, a significant proportion of the population remained parasitaemic, providing a potential reservoir for malaria transmission.
Jessica J. Briggs (Univerity of California San Francisco, USA) and her group sought to characterize malaria infections using amplicon deep-sequencing to longitudinally track P. falciparum clones within individuals with the goal of determining molecular force and duration of infection in the cohort. qPCR was performed on a blood sample taken every 28 days at routine visits as well as at non-routine visits during which malaria was diagnosed. All qPCR positive samples would be subject to a hemi-nested PCR to amplify a highly variable region of apical membrane antigen 1 (AMA-1), then use the bioinformatics pipeline SeekDeep to determine single-nucleotide polymorphisms (SNP) and haplotypes. Infection timeline plots were given for each individual to track the overall molecular force of infection (mFOI), which takes into account the number of new blood stage clones detected, number of susceptible people exposed, and duration of exposure. This statistic captures additional ongoing transmission that is not reflected by malaria incidence. The group found that both malaria incidence and mFOI increase after an increase of mosquito population. Interestingly, the study found that females clear infections faster than males across all age groups. School-aged children were found to have higher complexity infections, higher parasite density, and the longest duration of infection. Briggs discussed the usefulness of amplicon deep-sequencing in providing detailed information on within-host parasite dynamics, which can be used to estimate mFOI and duration of infection.
Chiara Andolina (Radboud University Nijmegen Medical Centre, Netherlands) presented on the kinetics of gametocyte production and human infectiousness to mosquitoes. The objectives of the study were to longitudinally quantify the contributions of different populations to the infectious reservoir through transmission assessments in the PRISM2 study cohort. The group tested individuals for malaria infection by microscopy, var gene acidic terminal sequence (varATS) qPCR, and gametocyte qRT-PCR. Participants who were qPCR positive the previous month or microscopy positive at the routine checkup were recruited for membrane feeding. Venous blood was drawn from 107 participants and used in a membrane feeding assay during which 60-80 mosquitos fed, then were dissected 10 days post-infection to assess the presence of oocysts. Only 7.2% of 538 total experiments resulted in infections. Adults had lower gametocyte densities than subjects of younger age. Symptomatic individuals were more infectious to mosquitoes compared to symptomatic ones. In a longitudinal assessment of infectiousness, it was found that 4 “superspreaders” were responsible for an astounding 62.6% of all infected mosquitoes. Andolina concluded that since asymptomatic super spreaders likely sustain transmission, identifying these populations is the key to controlling the spread of malaria and eliminating infectious reservoirs.
Symposium #122: The RTS,S Malaria Vaccine Pilot Implementation in Africa: Generating Data for Decision-Making
Public health specialists and scientists engaged in the RTS,S Malaria Vaccine Pilot Implementation Programme (MVIP) presented the key components of pilot evaluation activities that are generating data to inform a WHO policy recommendation on the broader use of RTS,S/AS01 in sub-Saharan Africa.
Mary Hamel (World Health Organization, Switzerland) opened the symposium with a review of the malaria context, noting the stall in global progress to reduce malaria illness and death and the need for new interventions to get malaria control back on track. Hamel summarized results from the RTS,S/AS01 Phase 3 trial (2009-2014) that confirmed the vaccine significantly reduces malaria incidence in children, including severe malaria. Three safety signals were identified during the trial whose causal relationship to the vaccine are unclear and may be chance findings – an excess in the number of meningitis cases, excess cerebral malaria cases, and, in post-hoc analysis, excess female deaths. The malaria vaccine pilot – recommended by WHO in 2016 – supports the pilot introduction of the malaria vaccine in routine childhood vaccination by the ministries of health in Ghana, Kenya and Malawi and the evaluation of the vaccine in routine use. The pilot evaluation will assess the programmatic feasibility of delivering a four-dose schedule, the vaccine’s impact on child mortality, and its safety in the context of routine use, with an emphasis on the safety signals seen in the phase 3 trial. Hamel noted that more than 1 million doses of vaccine have been administered and 430,000 children have received their first dose of the vaccine. An initial WHO recommendation on broader use of the vaccine may be considered as early as 2021, based on safety and impact data accrued in the pilot evaluation.
Rose Jalang’o (Ministry of Health, Kenya) presented the progress of the malaria vaccine pilot in Kenya, and a timeline of the Kenya MoH's consideration of the malaria vaccine over the last decade: from the formation of a national malaria vaccine subcommittee; the expression of interest by MoH to participate in the MVIP; the finalization of vaccine introduction plans; national regulatory authority approval of the vaccine for use in the pilots; vaccine introduction preparation, pilot initiation in 2019 and through the first year of implementation in 2020. Jalang’o highlighted some key challenges, including early misunderstanding among health workers of eligibility for the 4-dose vaccination schedule and how to catch up children who come late for vaccines; and, a decline in immunization coverage during the COVID-19 pandemic. Coordination and EPI support through remote means (virtual meetings) helped to mitigate the COVID-19 situation. She noted areas of pilot success: political and goodwill at all levels, strong MoH-partner collaboration, improved vaccine acceptability, mitigation strategies resulting in minimal disruptions during COVID-19, and strong county-level ownership of the pilot. Jalang’o noted some health system benefits of the pilot, including strengthening of pharmacovigilance systems, and more active collaboration between national immunization and malaria control programmes.
Sam Akech (KEMRI/Wellcome Trust, Kenya) described the sentinel hospital surveillance system, which is assessing safety signals identified in the Phase 3 trial, including meningitis and cerebral malaria, and the vaccine’s impact on severe malaria. Akech presented key elements of the evaluation, which builds on an established clinical information network (CIN) that supports hospitals to improve patient documentation and uptake of evidence-based pediatric clinical practice. Akech explained that the surveillance system in Kenya includes six public hospitals, located in different counties and serving children living in areas where RTS,S vaccine is offered and comparator areas, where the vaccine has not yet been introduced. A clinical protocol is established as part of the CIN, to guide clinicians on how to identify, diagnose, and manage patients. Some clinical equipment, materials and personnel were provided to hospitals in order to meet safety and impact objectives. Additional data quality assurance measures were introduced to ensure complete and high-quality data collection. Akech concluded that the CIN is an effective way to improve compliance with evaluation processes in a hospital-based study and that high-quality safety data to inform a recommendation on the wider use of RTS,S vaccine is being obtained.
Kwaku Poku Asante (Kintampo Health Research Centre, Ghana) described the community mortality surveillance system, another essential component of the malaria vaccine pilot evaluation. The approach is to collect data to evaluate the impact of the RTS,S vaccine on child mortality. Asante explained that existing mortality data sources (such as vital statistic systems) have limited and sometimes unreliable data. Evaluators aimed to augment and scale up the existing vital statistics infrastructure to collect reliable, timely and accurate mortality data to meet the evaluation objectives. Primary approaches included the mapping and sensitization of key mortality data stakeholders; training of health personnel and community volunteers to strengthen data collection; provision of logistical support for data collection; and development of a data management system that captures data on mortality. A quality assurance system is established and includes regular data reviews and visits to communities with no deaths reported. Successes of the programme were reported: the scaled system established a network of 7,000 community volunteers to identify and report deaths; 66 district health directorates are better able to conduct community mortality surveillance with a verbal autopsy, and there is improved data on vaccination status related to reported deaths. Surveillance was sustained throughout COVID-19, following national health and research guidelines.
Nicola Desmond (Malawi-Liverpool-Wellcome Trust, Malawi) presented an overview of the Health Utilization Study (HUS) that will evaluate the feasibility and community acceptability of the malaria vaccine pilot introduction in Malawi. Study questions include: how is RTS,S promoted and delivered through existing health systems; how do community members learn about and understand the vaccine; what social dynamics shape adoption of the 4-dose schedule; and, how does RTS,S uptake affect malaria prevention and treatment behaviors. The study methodology (observations, interviews, longitudinal design, and iterative approach) can help understand how to make RTS,S vaccine introduction feasible in communities. Study areas in Malawi encompass a mix of rural and peri-urban areas with varying levels of literacy, access to health services, and access to media and social media. Study teams explored knowledge of the vaccine, vaccine adherence, community engagement strategies, health worker training gaps, and how to integrate the vaccine with routine services. Desmond reported a generally positive response to RTS,S in Malawi, based on initial survey data. Primary caregiver exposure to RTS,S messages was at health facilities. Some confusion was reported among health providers and caregivers as to the age at which a child can receive the vaccine doses. Desmond noted that caregivers have generally high confidence in vaccines and health systems.
Paul John Milligan (London School of Hygiene and Tropical Medicine, United Kingdom) presented on the analysis approach for the Malaria Vaccine Pilot Evaluation (MVPE). The results of the RTS,S vaccine Phase 3 trial were reviewed, showing RTS,S vaccine safety and efficacy when provided in a 4 dose schedule, and the safety signals whose clinical significance was unclear. The objectives of MPVE include estimating the effect of RTS,S introduction on the incidence safety, impact and feasibility measures. The evaluation aims to assess the population effects of vaccine introduction in routine use, and since the expected impact or detectable safety measurements depends on vaccine coverage, the evaluation has been powered with that consideration. The analytic approach will compare the risk ratio of events of interest (e.g. meningitis, severe malaria) in children age-eligible to receive the RTS,S vaccine to children not age-eligible (older or younger) in vaccinating areas compared with the event risk ratios in comparator areas. This approach adjusts for differences between RTS,S vaccinating and comparator areas in access to a hospital; and the analysis does not require estimates of population denominators. It is anticipated that sufficient events will have accrued by 2021 for analysis of key safety outcomes and of the initial impact against severe malaria. A recommendation for broader vaccine use across sub-Saharan Africa could be made by WHO towards the end of 2021, if safety signals are satisfactorily resolved, and data indicate a positive impact. By the end of 2023, results will include coverage of the 4th dose, and impact on mortality.
Symposium #140: Spatial Intelligence to Optimise Public Health Interventions
Starting off the discussion, Anna Winters (Akros, Zambia) as symposium organizer explained the need to improve spatial intelligence in the fight against malaria, neglected tropical diseases, COVID-19 and others to achieve the SDGs as quickly as possible. She importantly noted that, despite having the most effective health treatments, high disease rates and mortalities were still being recorded majorly due to the gap in reaching target populations. It is important therefore to develop a better and more efficient system to ensure full coverage of disease interventions through the use of spatial intelligence.
Kafula Silumbe (PATH – MACEPA, Zambia) gave a presentation on “Spatial Intelligence for malaria elimination in Southern Province, Zambia” where he highlighted the use of the open-source platform called Reveal in identifying regions where mosquito control and malaria interventions could be effectively reached. The tool would be useful in optimizing indoor residual spraying (IRS) across the region. He pointed out that the IRS had previously faced several challenges including poor planning and budgeting and Reveal has been instrumental in curbing these setbacks. This was mainly due to the capacity of the Reveal tool to perform real-time monitoring and ultimately improve planning and decision-making in implementing malaria control strategies such as IRS. One of the main challenges faced by the program was the time-consuming programming and software development. Collaborations would therefore be important to improve the entire system’s efficiency in implementation. Plans for Reveal (2020-2021) are to implement IRS across seven districts in the region monitoring true coverage and redirecting resources as necessary through the support of the spatial intelligence platform. Finally, they plan to incorporate the use of the GRID3 datasets in the platform to allow mapping of areas that require interventions and estimating resources needed for the implementation.
Olatunde Adesoro (Malaria Consortium, Nigeria) shared his country’s experience from piloting Reveal for use in seasonal malaria chemoprevention (SMC) in Nigeria. SMC is the administration of full courses of antimalarial medication in areas of high seasonal transmission. This has been implemented since 2013 and ten million children have been covered in 2020. Challenges faced by SMC implementation include inaccurate coverage data and difficulty in reaching the aimed program coverage. The Reveal platform is expected to bring improvements through spatial mapping. Reveal deployment included a baseline assessment, usability testing, a pilot study, and operational research. Preliminary findings from usability testing showed the accuracy of data, easy monitoring and supervision, and easy drug reconciliation due to the ability to uniquely identify treatment history for children. The COVID-19 pandemic made most activities virtual so implementation and technical teams could not work together physically, but did so remotely. They also encountered internet connectivity issues in the field, security challenges, flooding, and bad terrain during the rainy season. Usability testing will guide the national malaria control programme on its uses for SMC.
Olena Borkovska (Columbia University, United States) in her talk titled “High-Resolution Population and Settlement Data for Impactful Malaria Interventions in Sub-Saharan Africa”, demonstrated GRID3’s implementation. The program aims to create efficient spatial data hence ensuring all areas are fully covered and development goals reached. This would be made possible by integrating enough details on pinpointing inhabitants in remote areas and utilizing this data to deliver resources and services for disease monitoring and control. Through the GRID3 project, Olena noted, high-resolution population maps are developed that would show the contrast in predicted population counts. This has been helpful for effective planning of malaria interventions in Zambia, under collaboration with Akros and the Ministry of Health. Objectives of GRID3 are to create planning maps and operationalize these with Reveal to create an implementation model and capacity building. This would ensure all remote areas are fully accounted for in service delivery, resources are efficiently distributed, and monitoring improvement of coverage from previous years. Future work will involve improving data accuracy, using Zambia as a reference point to expanding implementation of GRID3 workflows to other countries and integrating GRID3 into the Reveal tool in other countries.
Echoing the preceding presenters, Hugh Sturrock (Locational, United Kingdom) described the need to improve access to data and artificial intelligence to ultimately improve global health. Data availability has greatly improved with the development of programs and platforms to operationalize and visualize mapping data such as Reveal and DHIS2. Collaboration with data scientists would be useful for descriptive analysis and to extract the most relevant information. As an example, he pointed out that mapping malaria data fundamentally helps in designing disease prediction models and the effective distribution of disease-intervention resources across communities. To be able to understand and interpret this data, expertise in data interpretation would be a prerequisite. This could be quickly achieved via the use of algorithms-as-a-service such as locational.io. This organization builds and applies algorithms and proper tools, documentation, and expertise to enable quick interpretation of raw data by non-experts. This would play a key role in the speedy interpretation of data and implementation of disease-control strategies, thereby improving global health.
Symposium #163: Responding to the challenge of vector borne diseases in the context of urban expansion
Regina Rabinovich (MESA, ISGlobal, Spain), co-chair of the session together with Lee Hall (National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States), introduced the concept of the symposium by highlighting that when thinking about the urban context, the first challenge is how to define “urban”. She also noted that to address this complex landscape and its challenges, we will need to involve many different sectors that are not normally involved in vector control. Lee Hall noted that this area has received attention from the International Centers of Excellence for Malaria Research (ICEMR) programs, but further work is needed to better understand both risk and emerging interventions.
Peter Gething (Curtin University and Telethon Kids Institute, Australia) started his talk by saying that, according to the United Nations Development Programme (UNDP), by 2050 two thirds of the world’s population will live in urban areas. The implications of this urbanization process for malaria are still unknown. He then presented the work that the Malaria Atlas Program (MAP) undertook in an effort to answer this question: a descriptive analysis to discern the observable effects that urban areas are currently having in malaria transmission, and geostatistical modelling analysis to capture the current relationships between malaria prevalence and urbanicity and simulate the potential impact that urbanization may have in the future. Both analyses demonstrated malaria transmission and burden decline with increasing urbanicity, but Gething noted that caveats to this outcome are reliant on the accuracy of predicted trends for urbanization and continued relevance of present-day relationships.
The relationship between housing improvement and the built environment and vector control was discussed by Lucy Tusting (London School of Hygiene and Tropical Medicine, United Kingdom). The fact that the population in sub-Saharan Africa is predicted to double by 2050 creates a huge demand for new homes. Given the estimation that around 90% of malaria transmission occurs indoors, any features that block mosquitoes from entering the houses, such as closing eaves or screening windows and doors, can have a great impact. A systematic review published in 2015 concluded that improved housing could lower the risk of infection by 50%. Similarly, a survey across 29 national health systems in sub-Saharan Africa in 2017 saw 9-14% reductions in malaria linked with modern housing. Good housing also protects from other vector-borne diseases such as dengue, sleeping sickness or leishmaniasis. Tusting concluded by saying that Africa’s housing transition is a major opportunity, but we must link with sectors beyond health.
Abdisalan Noor (World Health Organization, Switzerland) started his talk by also asking the question ‘what is urban?’. As Rabinovich, he emphasized that a good definition becomes very important when we encounter huge differences between settings, cities and countries. When thinking about the urban malaria problem we are confronted with huge diversity, and some of the interventions that are normally applied to rural settings may not be useful for urban areas. He also posed additional questions that need to be addressed, such as the governance system in urban areas, the current and future of urban planning and sustainable living, and our current knowledge about urban health, health systems and service delivery in malaria-endemic countries. The answers to all these questions entail integrating malaria into surveillance and control of other vector-borne diseases, as well as characterizing and stratifying malaria risk in urban areas. The evidence to determine next steps in this area needs to be evaluated, and WHO is planning a series of consultations in the coming year.
Symposium #164: Integrating Functional, Population Genomic and Transcriptomic Data to Decipher Antimalarial Drug Resistance and Guide Drug Discovery
The employment of population genomic studies has led to discoveries of molecular markers and mechanisms underlying artemisinin resistance. The symposium began with Shannon Takala Harrison (University of Maryland School of Medicine, United States) showing that many genes identified in functional screens are supported by previous results from population genetic studies, including genes involved in endocytosis and proteasomal degradation. Harrison also showed that integrated selection of allele favoured by evolution (iSAFE) analysis could pinpoint favoured mutations within P. falciparum selective sweeps to identify new artemisinin resistance or compensatory mechanisms that could be validated by forwarding genetic approaches.
Resistance to antimalarial drugs, most recently to artemisinin drugs threatens malaria elimination and eventual global malaria eradication. Population genomics and transcriptomics have been used to identify regions of the parasite genome or gene expression patterns associated with both clinical and in vitro drug resistance phenotypes. Zbynek Bozdech (Nanyang Technological University, Singapore) presented a study on P. falciparum population transcriptomics in the context of evolving multidrug resistance in the Greater Mekong Subregion (GMS) to understand patterns that underlie artemisinin resistance in P. falciparum. A current study from the TRACII project from 15 different sites showed physiological relevance of artemisinin resistance-associated transcriptional profile (ARTP) including proteotoxicity, exported proteins, oxidative stress and gametocyte commitment. Bozdech showed that biological pathways linked to expression quantitative trait loci (eQTL) overlapped with ARTP. Remarkably, this led to a functional study of Cyclophilin B19 (CYP19B) which could drive significant resistance to artemisinin.
Jenna Oberstaller (University of South Florida, United States) presented recent findings that might explain how P. falciparum evolved resistance to artemisinins. A random, genome-scale subset of P. falciparum piggyBac-mutants selected from the saturation mutant-library was used to identify genes allowing parasites to survive the human fever. Pooled screening for mutants sensitive to heat shock enabled high-throughput characterization of gene function, identifying a number of genes involved in protein folding, vesicle-mediated transport, and host-cell remodelling driving the heat-shock response. Interestingly, apicoplast genes were highly responsive to febrile temperatures. Connecting these findings to previous and concurrent studies, the apicoplast isoprenoid biosynthesis pathway drives vesicular trafficking, which plays a critical role in host-cell remodelling, haemoglobin transport and digestive vacuole function. Interestingly, this study further linked heat-shock response to artemisinin resistance, leading to the integrative hypothesis that pathways gained through the algal endosymbiont-derived apicoplast allowed parasites to survive host fever—and they are now being utilized by the parasite to survive artemisinin treatment.
Stanley C. Xie (University of Melbourne, Australia) began his presentation by giving an overview of our current understanding of the mechanisms of artemisinin resistance and mode of action that are still under debate. From recent studies, dihydroartemisinin (DHA) has been shown to induce oxidative stress and proteotoxicity, leading to parasite death. This study led to the evaluation of proteasome inhibitors as promising potent antimalarials. In corroboration with Takeda and MMV, proteasome inhibitors were further examined by a hit-to-lead program where MPI-5 was found to be efficacious and well-tolerated in mice. Cryo-EM was used to solve the fine structure of the Pf 20S (Proteasome subunit) and reveal its interactions with inhibitors. He noted that this advanced technology could be used to guide rational drug discovery.
Symposium #166: Current Knowledge of Mosquito-Stage Malaria Parasite Biology: Implications for Developing a Robust in vitro Culturing System
During this session, the Co-Chair Flaminia Catteruccia (Harvard TH CHAN, USA) questioned the panellists on why we do not have a reliable in vitro system yet and what we could achieve if we had one. Potential answers to these questions were brought by the speakers.
Shirley Luckhart (University of Idaho, USA) reported on host factors in the blood meal as regulators of vector-parasite interaction and transmission in malaria. She addressed questions on why mosquitoes feed on blood, which constitutes a high risk, but also a high payoff. If there were more to it than reproduction, this characteristic could be used to prevent malaria parasite infection of and transmission by mosquitoes. A mosquito blood meal contains pathogen-derived factors as well as nutrients. Luckhart talked about insulin and mitogen-activated protein kinase (MAPK) signalling as ancient regulators of homeostasis in invertebrate and the insulin/insulin-like growth factor signalling cascade components controlling the biosynthesis of coenzyme A (CoA). Their findings showed that P. falciparum activates c-Jun N-terminal kinase (JNK) signalling to inhibit mosquito use of pantothenate, increasing available substrate to its own CoA biosynthesis. Luckhart suggested the exploitation of blood as a source of signalling mediators.
Marcelo Jacobs-Lorena (Johns Hopkins Bloomberg School of Public Health, USA) discussed the gametocyte-to-oocyst transition as a most valuable target for setting-up the Plasmodium in vitro system. One of his key points was that malaria did not exist without the mosquito vector. An understanding of the development of Plasmodium parasites in mosquito vectors is needed, especially in the midgut which seemed to be the best place to target the parasite in the mosquito due to the existence of an "oocyst bottleneck". The team designed a plasminogen activation inhibitor (PAI) which could selectively inhibit tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) that are required for ookinete formation. Through the fibrinogen/fibrin network of the mosquito midgut blood bolus, fibrinogen promotes red blood cell (RBC) aggregation and could be useful in inhibiting midgut development of P. falciparum. These findings could be translated to the field by introducing transgenic mosquitoes expressing huPAI. As the P. berghei in vitro development from gametocyte to ookinete is well established, this knowledge could help the development of an in vitro system for high-throughput screening for P. falciparum.
Ashley M. Vaughan (Seattle Children’s Research Institute, USA) presented on "Take it and make it: oocyst development required nutrients uptake and de novo biosynthesis" with emphasis on oocyst development and generation of infectious sporozoites. They knocked out the Abcb5 transporter in P. berghei and found that oocyst development was delayed and no sporozoites were produced and linked this phenotype to a possible role of Abcb5 in the uptake of mosquito lipids. He also addressed the question of whether parasites make their own nutrients presenting published data on the P. falciparum fatty acid synthase (FAS) II, which is essential for oocyst maturation and the knockout of which produces oocysts that mature but fail to form. P. falciparum oocyst development, therefore, requires uptake of blood meal-associated nutrients and the de novo synthesis for sporozoite production. Oocysts appear to sense nutrient levels and can enter a dormant state. He concluded by pointing out the fact that in vitro sporozoite production is possible but still challenging.
Photini Sinnis (Johns Hopkins Bloomberg School of Public Health, USA) explained how sporozoite migration from mosquito to mammalian host is associated with a series of bottlenecks, including midgut phase, skin phase, blood phase and liver phase. Quantitative approaches such as the Ross-MacDonald Model could help in understanding mammalian malaria transmission and the probability that a sporozoite-carrying mosquito will feed upon and infect another person resulting in malaria infection. The use of the P. yoelii/mice combination already showed that infection likelihood depends on the sporozoite load in the mosquito salivary glands. Interestingly, blood meal acquisition has no impact on the likelihood that an infected mosquito will initiate malaria transmission. Even though sporozoite-based vaccines have been the only ones to show some efficacy in the field, uncertainties concerning the skin phase of Plasmodium infection persist. An in vitro system for producing human-infectious sporozoites would be a huge advance to these efforts.
Symposium#167: Tracking the Threat of pfhrp2/3 Gene Deletions and Future Alternatives to HRP2-based Malaria Diagnosis
P. falciparum parasites with histidine-rich protein 2 and 3 (hrp2/3) gene deletions are a growing problem, diminishing the performance of HRP2-based RDTs currently used for malaria case management and surveillance in affected areas. The symposium provided a comprehensive overview with presentations on the global distribution, country experiences on high throughput screening, hrp2/3 deletion characterization and reporting, as well as alternative diagnostic tests.
Jane A. Cunningham (World Health Organization, Switzerland) spoke about how PfHRP2/3 gene deletions are actually being tracked by the WHO. A majority of malaria rapid diagnostic tests (RDTs) target PfHRP2 because of its stability. PfHRP3 is another protein that shares some epitopes with HRP2, which allows antibodies on some brands of RDTs to bind HRP3. This is important for detection of malaria parasites that have HRP2 deletions. The first clinical isolates lacking PfHRP2/3 were found in Peru, but have since been found across the globe. In response to these findings, WHO built an international network of laboratories to assist countries in identifying, tracking, and mapping parasites with gene deletions. Another impressive development was the WHO Malaria Threat Maps, an open source tool for interactive data visualization of global malaria threats such as drug resistance and gene deletions. Data is extracted from published reports and maps are updated every 4-6 weeks. However, Cunningham pointed out that the original source for each data point is critical for properly interpreting these data. In a systematic review of 38 publications, only 5% of studies met the 7-point criteria for accurate investigation and reporting of deletions. Therefore, in order to plan appropriate interventions and decrease morbidity and mortality, the quality of many surveillance studies must be improved.
Eric Rogier (Centers for Disease Control and Prevention, United States) began his presentation by providing a review of malaria rapid diagnostic tests (RDTs), which mainly probe for histidine-rich protein 2/3 (HRP2/3) and/or lactate dehydrogenase. Diagnosis by RDT involves assessment of the absence or presence of target proteins, with multi-target RDTs sometimes providing combinations of positives and negatives that make diagnosis more challenging. Rogier presents a different method for screening parasites lacking HRP2/3 which starts with a phenotypic screening using a bead-based multiplex antigen detection panel, followed by molecular characterization using PCR. Multiple sample types can be screened for Plasmodium antigens by using antibody-bound beads to capture the corresponding antigen and detect it with a fluorescently labelled antibody. Using a phenotypic screen is advantageous because it can be used in a high-throughput format, is relatively inexpensive, detects the same proteins as RDTs, and can specify those samples that truly require further molecular characterization. Addition of targets to a hypothetical multiplex panel can expand the potential for interpreting phenotypes and better describe an individual’s Plasmodium infection status.
Jonathan B. Parr (University of North Carolina, United States) presented “The evolving approach to pfhrp2/3 deletion characterization”. Confirming the absence of a gene is not straightforward and several steps and methods are required to make a deletion call. Initial evidence can be obtained via microscopy, RDT and PCR, whereas for confirmatory evidence multiple PCRs are required per current guidelines. However, even with these approaches, it remains challenging to be confident in the absence of a gene. Parr gave an example from work in the Democratic Republic of the Congo (DRC), where they conducted multiple PCR assays and identified eight individual deletions using an established PCR-based approach. However, whole-genome sequencing indicated intact genes and a bead-based antigen screen confirmed circulating antigen, leading to the conclusion that the false-negative RDT results among symptomatic subjects in DRC were not due to deletions. This example demonstrated the value of using multiple approaches to overcome weaknesses of single methods, and for improved rigor. During the talk, Parr shared three key PCR workflow changes they made that improved their performance (maximizing DNA template, using AmpliTaq Gold polymerase and using additional cycles (>45) of amplification). He further briefly described new emerging technologies (molecular inversion probe (MIP), Oxford nanopore technologies, and digital droplet PCR). He shared a recent example of hrp2/3 MIP sequencing applied during a large cross-sectional survey, which enabled deletion calls, visualization of deletion breakpoint regions in the parasite’s chromosomes, and initial assessment of the evolutionary forces impacting parasites in the regions sampled. He closed with a reference to a WHO reference laboratory network that is available to support hrp2/3 deletion surveillance.
Dionicia Gamboa (Universidad Peruana Cayetano Heredia, Peru) presented options, performance and affordability of promising alternatives to HRP2-based rapid tests. Alternative tests include automated microscopy and cell phone-adapted systems for parasite detection, ultrasensitive RDTs, multiplex immunoassays, and high-throughput screening systems for antigen detection, loop-mediated isothermal amplification (LAMP) for DNA detection, and urine or saliva samples as non-invasive methods. Automated microscopy, tested in a study in Peru, was found to have the same performance as microscopy by trained professionals, however, it requires good quality of the blood slides. LAMP, also tested in Peru, identified four times more individual infections than microscopy, whereas sample preparation and storage requirements outweighted the advantages. Multiplex immunoassays were useful for identifying hpr2/3 deletions and play an important role in the development of ultra-sensitive RDTs. The immunoassays use both frozen blood samples and dried blood spots. The development of new RDTs and accuracy trials, with genomic characterization for hrp2/3-deleted samples, is part of Gamboa’s ongoing work. Moreover, new biomarkers used for sero-surveillance are promising but need further validation (glutamate dehydrogenase, MSP10).
This report is brought to you by the MESA Correspondents. Senior editorial support has been facilitated by the Organizers and Co-Chairs of the symposia, Valentina Mangano (University of Pisa) and Julie Chaccour (Independent Consultant). This report is cross-posted on the MESA website and on MalariaWorld.