Daniel E. Neafsey

Early-phase trials of candidate malaria interventions in endemic areas often use “time-to-first” infection as a primary efficacy endpoint. This approach does not assess infection risk after the initial infection, and its censoring of trial participants after their first infection can introduce bias by removing more high-risk participants from control than treatment arms over time. Efficacy estimate precision may also decline as risk set size decreases. Here we assessed the utility of genotyping all infections in an intervention trial to determine the total infection incidence in each participant for the full follow-up period. Specifically, we genotyped all Plasmodium falciparum infections detected in a previously-reported phase 2 trial of the antimalarial monoclonal antibody CIS43LS in 330 Malian adults. We determined the molecular force of infection (molFOI; rate of new P. falciparum strains) for each participant, expressing efficacy as the percent molFOI reduction in CIS43LS versus placebo arms (Eff _molFOI ). Six-month Eff _molFOI was 86.5% (95% CI: 80.1-90.8) and 70.7% (61.0-78.0) for the 40 mg/kg and 10 mg/kg doses, respectively. These estimates are similar to those from time-to-first infection analysis, but more precise when analyzing efficacy by trial-month and when stratifying by P. falciparum infection-status at enrollment. Participants infected at enrollment had lower Eff _molFOI and acquired infections earlier in the trial, creating systematic differences in risk composition between arms over time. Genotyping showed no evidence for escape-polymorphisms in the CIS43LS epitope. These results demonstrate how genotypic endpoints can provide a more comprehensive understanding of candidate malaria interventions. One sentence summary: Malaria parasite genotyping and molecular force of infection inference improves standard malaria intervention efficacy analysis.

Malaria-causing Plasmodium parasites are transmitted between vertebrate hosts via diverse species of Anopheles mosquitoes. The P47 protein in Plasmodium falciparum has been shown to affect transmission in various anopheline species by mediating evasion of mosquito innate immunity in an allele-specific manner. The Pfs47 gene that encodes P47 exhibits high genetic divergence across continents, as well as high genetic diversity within many parasite populations, but the impact of polymorphisms on transmission success in different sympatric vectors has not been previously studied in a field context. Here we report parasite genotyping data from naturally infected, field-collected mosquitoes at sites harboring at least two prevalent sympatric vector species, targeting Pfs47 in addition to the CSP , AMA1 , SERA2 , and TRAP parasite antigens. Collections took place at Ndangueng, Cameroon and Benguia, Gabon between November 2019 and July 2021, yielding infected Anopheles gambiae at both sites in addition to Anopheles moucheti in Cameroon and Anopheles funestus in Gabon. We found no significant difference in the mean number of parasite strains in infected sympatric mosquito species from each site. However, Pfs47 exhibited a significant difference in the prevalence of an amino acid polymorphism (I248L) between An. moucheti and An. gambiae in Cameroon. Polymorphisms at two additional positions (N272Y/I and P369H) displayed significant linkage disequilibrium with I248L, and there were significant differences in the distribution of Pfs47 haplotypes between sympatric mosquito species at both sites. These findings demonstrate divergent selection on P. falciparum mediated by sympatric vector species, suggesting that parasite genotype significantly impacts local vectorial capacity by modulating species-specific vector competence.

A central challenge in infectious disease biology is anticipating whether pathogen proteins targeted by vaccines, drugs, or diagnostics can evolve in ways that reduce intervention efficacy. Evolutionary constraint is therefore relevant to selecting targets likely to remain effective after deployment. Yet low diversity in contemporary pathogen populations is often interpreted as evidence of evolutionary constraint, an inference that can be misleading. In this Personal View, we use Plasmodium falciparum malaria to illustrate how contemporary population diversity and longer-term cross-species divergence can inform assessments of evolutionary constraint. A severe population bottleneck reduced diversity across the P. falciparum genome, meaning that low diversity in a particular protein-coding gene does not necessarily indicate unusual constraint. We place gene-level diversity in its genome-wide context, compare it with divergence across related ape-infecting malaria parasites, and examine how both relate to experimental essentiality. We then examine diversity and divergence along vaccine, drug, and diagnostic targets, considering them in relation to known resistance-associated mutations and other routes of evolutionary escape. We introduce PEEP (Protein Evolution Explorer for Plasmodium), a public interactive resource for exploring diversity and divergence across P. falciparum protein-coding genes and along individual proteins. Although our analyses focus on P. falciparum malaria, the implications extend across pathogens: low diversity alone is insufficient evidence of evolutionary constraint, and assessments of intervention durability should consider constraint alongside potential routes of evolutionary escape.

Genomic tools are increasingly important for conservation and wildlife health surveillance, yet their use is often constrained by limited resources, sample quality, and the need for minimally invasive approaches. Here we show that untargeted low-coverage sequencing applied to scale-clip DNA can jointly address population structure, genotype–phenotype associations, and pathogen surveillance in timber rattlesnakes (Crotalus horridus), a species threatened by habitat fragmentation, human encroachment, and the emerging fungal pathogen Ophidiomyces ophidiicola. We sequenced 107 snakes sampled from eight populations in the Appalachian Mountains of the northeastern United States to a mean depth of 0.94x per individual. Population differentiation is pronounced and only weakly correlated with geographic distance, and inbreeding coefficients in some populations are high enough to suggest fitness consequences. Genome-wide association analysis identifies a locus for the black-to-yellow color morph containing ALDH4A1, a member of a gene family implicated in vertebrate pigmentation but distinct from melanin pathway pigmentation genes. Our untargeted sequencing method also captures host-associated microbes: O. ophidiicola loads are higher in snakes with clinical signs of snake fungal disease, though some asymptomatic individuals carry substantial loads, and skin-associated microbial communities are disrupted in snakes with higher fungal burdens. That all of these inferences derive from the same minimally invasive sampling and sequencing workflow highlights the scalability of this approach for biodiversity conservation.

Sporozoites, the infectious stage of Plasmodium malaria parasites, must invade two different tissues in two very different hosts to ensure transmission: the mosquito salivary glands and the human liver. Whether infectivity to hepatocytes is affected by physiological components, such as the availability of nutrients within the Anopheles female, is not well understood. Here we show that Plasmodium falciparum sporozoite infectivity to hepatocytes is increased if mosquitoes take an additional blood meal while oocysts are developing. Infection rates of HC04.J7 hepatocytes at 24 h were 1.8-fold higher for P. falciparum salivary gland sporozoites isolated from Anopheles gambiae females fed twice (2BF) compared to those which only received the infectious blood meal (1BF). This increase in infectivity was observed at an early infection timepoint (day 11) and was accompanied by a marked shift in the transcriptional profiles of 2BF salivary gland sporozoites, identifying genes likely involved in infection and development in the mammalian host. Overall, our data reveal new factors potentially involved in sporozoite infectivity, and have broad implications for understanding transmission of malaria in endemic areas where multiple blood meals commonly shape available resources for adult female mosquitoes.

Both vaccination and seasonal chemoprevention are effective interventions against malaria, but both morbidity and mortality due to malaria remain high, particularly in sub-Saharan Africa. A recent clinical trial showed that the combination of these interventions provided substantial protection against malaria over a five-year period. We performed amplicon-based genotyping of highly polymorphic parasite antigens within 1530 samples from this clinical trial. We evaluated the complexity of infection within these samples, finding that participants who received both interventions had less complex infections (fewer genetically distinct parasite strains) than participants who received either intervention given alone. We also evaluated the prevalence of parasites matching the vaccine construct CSP, at the level of haplotypes, epitopes, and individual amino acid changes. We found no significant differences in the prevalence of vaccine-matching alleles between vaccinated and unvaccinated groups. For context, this study genotyped about half as many non-vaccinated participants as the original phase 3 trial for RTS,S. While a comparable number of vaccinated individuals were genotyped in both studies, the infections in this study had less complex infections, leading to an overall lower number of parasite strains to analyze. These results suggest that the combination of vaccination with seasonal chemoprevention provides protection from infection, in addition to clinical disease, and they highlight the importance of continued molecular surveillance as malaria interventions are deployed.

Vector-borne diseases (VBDs) are a leading cause of morbidity and mortality worldwide. Antigens driving host immune responses to pathogens and vectors can serve as vaccine candidates and biomarkers of previous exposure, but the immunogenicity of relevant proteomes remains undercharacterized. To comprehensively profile antibody responses to VBDs and the vectors themselves, we developed a highly multiplexed phage display library, VectorScan, containing more than 250,000 peptides derived from diverse arthropod vectors and prevalent vector-borne pathogens. We used phage immunoprecipitation sequencing to screen VectorScan against blood samples with experimental and natural exposures to arboviruses, malaria parasites, and mosquitoes. We analyzed quantitative measurements of peptide seroreactivity to identify epitopes driving viral serotype-level exposure signatures and mosquito sialome antigens. Mosquito-directed antibody responses were quantitatively associated with natural viral and parasite exposure. We leverage VectorScan to define the relationship between the intensity of mosquito exposure and the rate of malaria infection in children, demonstrating the public health utility of a tool for paired vector and pathogen serosurveillance.

Therapeutic efficacy studies (TESs) are the standard to evaluate antimalarial drug efficacy and guide malaria treatment policy. TESs are particularly relevant now, with resistance to first-line regimens emerging in sub-Saharan Africa. For TESs, a range of parasite genotyping and data analyses are available for genotype correction, a process to distinguish whether recurrent parasitemia after therapy is due to recrudescence of initially infecting parasites (treatment failure) or a new infection. The choice of methods for laboratory genotyping and data analyses can have a large effect on how outcomes are classified, and thereby on trial results. The currently recommended and most widely used laboratory and analytical methods for TES genotyping do not incorporate recent methodological advances and can produce biased results. As such current TES results can be difficult to interpret, especially in areas with high malaria transmission, such as much of sub-Saharan Africa. Thus, improving the accuracy and reliability of TES genotyping and data analysis are a major priority. To that end, we present target product profiles that outline key specifications for genetic data generation, processing, and data analysis, with the goal of creating rigorous and consistent community standards. Primary recommended specifications for laboratory methods include high sensitivity, specificity, and reproducibility, and guidance on the number and genetic diversity of targets; criteria which are best and likely only met by amplicon sequencing. Primary recommendations for data analysis methods include high classification accuracy, accounting for errors in genotyping, and accounting for alleles matching by chance. All laboratory and data analysis methods used should be systematically validated and publicly documented so that TES results, which have major policy implications, can be relied upon for sound programmatic decision making.

Species distributed across heterogeneous environments often evolve locally adapted populations, but understanding how these persist in the presence of homogenizing gene flow remains puzzling. In Gabon, Anopheles coluzzii, a major African malaria mosquito, is found in various ecological settings, including urban areas, remote rural villages, and forested environments away from any human presence. This study investigates the genomic signatures of local adaptation in populations from distinct environments including the urban area of Libreville, and two proximate sites 10 km apart in the La Lopé National Park (LLP), a village and its sylvatic neighbourhood. Whole genome re‐sequencing of 96 mosquitoes unveiled 5.9 million high‐quality single nucleotide polymorphisms. Coalescent‐based demographic analyses suggest an ∼ 12,000‐year‐old divergence between Libreville and La Lopé populations, followed by a secondary contact ( ∼ 4000 ybp) resulting in asymmetric effective gene flow. The urban population displayed reduced effective size, evidence of inbreeding, and strong selection pressures likely associated to insecticides or pollution present in urban settings, as suggested by the hard selective sweeps detected in genes involved in detoxification and insecticide resistance. In contrast, the two geographically proximate LLP populations showed larger effective sizes, and distinctive selective signals, notably soft‐selective sweeps on the standing genetic variation. Although presumably neutral loci failed to discriminate between LLP populations, our findings support that microgeographic adaptation can swiftly emerge through selection on standing genetic variation despite gene flow. This study contributes to the growing understanding of evolution of populations in heterogeneous environments amid ongoing gene flow and how major malaria mosquitoes adapt to humans and its environment.

Malaria in South America remains a serious public health problem. Anopheles ( Nyssorhynchus ) darlingi is the most important malaria vector across tropical Latin America. Vector-targeted disease control efforts require a thorough understanding of mosquito demographic and evolutionary patterns. We present and analyze whole genomes of 1094 An. darlingi (median depth 18x) from six South American countries. We observe deep geographic population structure, high genetic diversity including 13 putative segregating inversions, and no evidence for sympatric cryptic taxa despite high interpopulation divergence. Strong signals of selection are plausibly driven by insecticides, especially on cytochrome P450 genes. Our results will facilitate effective mosquito surveillance and control while highlighting ongoing challenges that a diverse vector poses for malaria elimination in the Western hemisphere.