Publications

Influenza viruses present a significant public health risk, causing substantial illness and death in humans each year. Seasonal flu vaccines must be updated regularly, and their effectiveness often decreases due to mismatches with circulating strains. Furthermore, inactivated vaccines do not provide protection against shifted influenza viruses that have the potential to cause a pandemic. The highly pathogenic avian influenza H5N1 clade 2.3.4.4b is prevalent among wild birds worldwide and is causing a multi-state outbreak affecting poultry and dairy cows in the United States (US) since March 2024. In this study, we have generated a NS1 deficient mutant of a low pathogenic version of the cattle-origin human influenza A/Texas/37/2024 H5N1, namely LPhTXdNS1, and validated its safety, immunogenicity, and protection efficacy in a prime vaccination regimen against wild-type (WT) A/Texas/37/2024 H5N1. The attenuation of LPhTXdNS1 in vitro was confirmed by its reduced replication in cultured cells and inability to control IFNβ promoter activation. In C57BL/6J mice, LPhTXdNS1 has reduced viral replication and pathogenicity compared to WT A/Texas/37/2024 H5N1. Notably, LPhTXdNS1 vaccinated mice exhibited high immunogenicity that reach its peak at weeks 3 and 4 post-immunization, leading to robust protection against subsequent lethal challenge with WT A/Texas/37/2024 H5N1. Altogether, we demonstrate that a single dose vaccination with LPhTXdNS1 is safe and able to induce protective immune responses against H5N1. Both safety profile and protection immunity suggest that LPhTXdNS1 holds promise as a potential solution to address the urgent need for an effective vaccine in the event of a pandemic for the treatment of infected animals and humans.

A multistate outbreak of highly pathogenic avian influenza virus (HPAIV) H5N1 in dairy cows was first reported on March 25, 2024, in the United States (US), marking the first discovery of HPAIV H5N1 in cattle. Soon after, a dairy worker on an affected dairy farm became the first human case linked directly to this outbreak. Studies with influenza A virus (IAV) require secondary methods to detect the virus in infected cells or animal models of infection. We modified the non-structural (NS) genome segment of the human A/Texas/37/2024 (HPhTX) H5N1 virus to create a recombinant virus expressing nanoluciferase (HPhTX NSs-Nluc), enabling the tracking of virus in cultured cells and mice via in vitro, ex vivo, and in vivo imaging systems (IVIS). In vitro, HPhTX NSs-Nluc showed growth and plaque characteristics similar to its wild-type (WT) counterpart. In vivo, HPhTX NSs-Nluc allowed tracking viral infection in the entire animals and in the organs of infected animals using in vivo and ex vivo IVIS, respectively. Importantly, the morbidity, mortality, and replication titers of HPhTX NSs-Nluc were comparable to those of the WT HPhTX. In vitro, HPhTX NSs-Nluc was inhibited by Baloxavir acid (BXA) to levels observed with WT HPhTX. We also demonstrate the feasibility of using HPhTX NSs-Nluc to evaluate the antiviral activity of BXA in vivo. Our findings support that HPhTX NSs-Nluc represents an excellent tool for tracking viral infections, including the identification of prophylactics or therapeutics for the treatment of the HPAIV H5N1 responsible of the outbreak in dairy cows.

Malaria parasites are obligately sexual hermaphrodite protozoans with gamete fusion occurring in the mosquito midgut, followed by meiosis and recombination. Malaria parasite populations show a spectrum of populations structures ranging from predominantly selfing to highly outcrossed. The fitness consequences of selfing and outcrossing for malaria parasites are poorly understood: this project was designed to investigate the dynamics of gamete fusion within the mosquito midgut and the relative fitness of selfed and outcrossed zygotes. We generated florescent-labelled clones of NF54 (mCherry), an African parasite, and NHP4026 (GFP), a Thai parasite, crossed these parasites, and scored genotypes of 8540 oocysts from 435 mosquitoes sampled from 7 to 14 days post infection. We observed decreasing proportions of outcrossed oocysts and increasing levels of inbreeding over the course of the infection in two independently replicated crosses. These results are consistent with the faster maturation of transmissible sporozoites derived from outcrossed compared with selfed oocysts. Our results suggest a substantial outcrossing advantage, perhaps because this allows for the removal of deleterious mutations accumulated during asexual parasite replication in the vertebrate host. We also found that selfed NF54 oocysts were larger than outcrossed or selfed NHP4026 oocysts, which may influence production of sporozoites and onward transmission. We conclude that fluorescent labelled parasites provide clear resolution of mating patterns, temporal dynamics and transmission potential of malaria parasites in mosquitoes. Importantly, faster maturation of outcrossed parasites can maximize levels of recombination in transmitted malaria parasite populations.

BACKGROUND: The microbiome of disease vectors can be a key determinant of their ability to transmit parasites. Conversely, parasite infection may modify vector microbiomes. We explore the interactions between the Biomphalaria glabrata snail microbiome and the blood fluke Schistosoma mansoni, responsible for an estimated 200,000 human deaths each year. We have previously shown that the snail hemolymph (i.e. blood) and organs harbor a diverse microbiome. Here, we investigate the impact of schistosome infection on snail microbiomes, hypothesizing that invading schistosomes can alter the snail microbiomes in both composition and abundance over the course of infection, as developing schistosome parasites are in close contact with the host tissues.

METHODS: We generated cohorts of uninfected and S. mansoni-infected snails. We collected snail hemolymph and hepatopancreas (i.e. liver) at eight timepoints during the pre-patent and patent periods of schistosome infection. We quantified bacterial density using qPCR and profiled the microbiome composition of all samples by sequencing the V4 region of the 16S rRNA.

RESULTS: Surprisingly, schistosome infection had no effect on bacterial density and limited effect on the microbiome composition, affecting mainly the hemolymph during the pre-patent period (at days 7 and 21). Organ and hemolymph microbiomes were relatively stable over time for both infected and uninfected snail cohorts. The sample type (hemolymph, hepatopancreas) was the major driver of the differences observed in microbiome composition.

CONCLUSIONS: The limited impact of schistosome infection on the host snail microbiomes might be explained by the long-term interaction of the two partners. Further investigations into the interactions among snails, their microbiomes and schistosome parasites may suggest strategies to disrupt the parasite lifecycle and, consequently, schistosomiasis transmission.

The malaria elimination programme in Kayin State (Myanmar) uses malaria posts for rapid detection and treatment, together with mass drug administration in high-transmission villages, which has reduced transmission by 97%. Here we examine the impact of control on parasite genomic parameters to inform future control efforts. Using 2,270 genome-sequenced Plasmodium falciparum infections from 283 malaria posts, sampled over 58 months (2015-2020), we find that parasite effective population size decreased over the study period, but there was minimal change in artemisinin resistance allele frequency until 2020, when just one predominant genotype (carrying kelch13-R561H) remained. We observed sustained localized transmission of unique parasite genotypes revealing transmission chains and positive correlations in parasite relatedness for ≤20 km. Mass drug administration resulted in parasite founder effects, providing genomic evidence for the efficacy of this control tool. These results reveal changes in population structure driven by control and rapid shifts in allele frequency in a parasite population close to elimination.

Genetic markers for detecting hybridization and measuring population genetic parameters must be informative and cost-effective. Most population genetic studies within the Schistosoma haematobium species group rely on either a two-marker system consisting of the mitochondrial cytochsniromeoxidase 1 (cox1) and the nuclear internal transcribed spacer (ITS) markers or, at the other extreme, millions of single nucleotide variants (SNVs) from whole genome/exome sequencing. cox1 and ITS studies contain minimal population genetic information, but whole genome sequencing is cost-prohibitive. We examined approximately 38 million previously published, whole genome SNVs genotyped in 162 S. haematobium and Schistosoma bovis sampled across Africa. We compared population genetic parameters from 4000 panels of 10-100 000 randomly sampled SNVs to results from the whole genome dataset to test the resolution of reduced representation sequencing in schistosomes. We found that panels of 500 SNVs captured >99% of the population genetic information contained in the whole genome dataset by using Procrustes transformed principal component analyses and ancestry estimates (r² = 0.85). Additionally, the costs of genotyping parasites with an amplicon panel are two to three times less than whole genome sequencing. Our results show that moderately sized amplicon panels targeting random SNVs provide an efficient approach to large-scale, field-based schistosome surveillance. This article is part of the Royal Society Science+ meeting issue 'Parasite evolution and impact in action: exploring the importance and control of hybrid schistosomes in Africa and beyond'.

The relationship between parasite genotype and pathogenesis is largely unknown for Cryptosporidium, a leading cause of diarrheal disease in children. An array of parasites with similar genomes produces varied disease outcomes in different hosts. Here, we isolate and characterize Cryptosporidium parvum strains that show marked differences in virulence and persistence in mice. Taking advantage of the sexual lifecycle of this eukaryotic pathogen, we use genetic crosses to discover the underlying chromosomal loci. Whole-genome sequencing and bulk segregant analysis of infection selected progeny mapped three loci on chromosomes 2, 6, and 7 associated with the ability to colonize and persist in mice and the positions of drug resistance genes. The chromosome 6 locus encodes the hyper-polymorphic surface glycoprotein GP60. Reverse genetic studies in both parental strains demonstrate that GP60 controls parasite burden and virulence, but not persistence, and reveal the dominance of the less virulent allele, suggesting it restricts virulence.

Pooled sequencing provides a rapid cost-effective approach to assess genetic variation segregating within populations of organisms. However, such studies are typically limited to single nucleotide variants and small indels (≤ 50bp), and have not been used for structural variants (SVs; >50bp) which impact large portions of most genomes and may significantly impact phenotype. Here, we examined SVs circulating in five laboratory populations of the human parasite Schistosoma mansoni by generating long-read sequences from pools of worms (92 -152 per population). We were able identify and genotype 17,446 SVs, representing 6.5% of the genome despite challenges in identifying low frequency variants. SVs included deletions (n=8,525), duplications (n=131), insertions (n=8,410), inversions (n=311), and translocations (n=69) and were enriched in repeat regions. More than half (59%) of the SVs were shared between ≥4 populations, but 12% were found in only one of the five populations. Within this subset, we identified 168 population-specific SVs that were at-or-near fixation (>95% alternate allele frequency) in one population but missing (<5%) in the other four populations. Five of these variants impact the coding sequence of 6 genes. We also identified 8 SVs with extreme allele frequency differences between populations within quantitative trait loci for biomedically important pathogen phenotypes (drug resistance, larval stage production) identified in prior genetic mapping studies. These results demonstrate that long-read sequence data from pooled individuals is a viable method to quickly catalogue SVs circulating within populations. Furthermore, some of these variants may be responsible for, or linked to, regions experiencing, population-specific directional selection.

The human parasitic fluke, Schistosoma haematobium hybridizes with the livestock parasite S. bovis in the laboratory, but the frequency of hybridization in nature is unclear. We analyzed 34.6 million single nucleotide variants in 162 samples from 18 African countries, revealing a sharp genetic discontinuity between northern and southern S. haematobium. We found no evidence for recent hybridization. Instead the data reveal admixture events that occurred 257-879 generations ago in northern S. haematobium populations. Fifteen introgressed S. bovis genes are approaching fixation in northern S. haematobium with four genes potentially driving adaptation. We identified 19 regions that were resistant to introgression; these were enriched on the sex chromosomes. These results (i) suggest strong barriers to gene flow between these species, (ii) indicate that hybridization may be less common than currently envisaged, but (iii) reveal profound genomic consequences of rare interspecific hybridization between schistosomes of medical and veterinary importance.

There are limited control measures for the disease schistosomiasis, despite the fact that infection with parasitic blood flukes affects hundreds of millions of people worldwide. The current treatment, praziquantel, has been in use since the 1980’s and there is a concern that drug resistance may emerge with continued monotherapy. Given the need for additional antischistosomal drugs, we have re-visited an old lead, meclonazepam. In comparison to praziquantel, there has been relatively little work on its antiparasitic mechanism. Recent findings indicate that praziquantel and meclonazepam act through distinct receptors, making benzodiazepines a promising chemical series for further exploration. Previous work has profiled the transcriptional changes evoked by praziquantel treatment. Here, we examine in detail schistosome phenotypes evoked by in vitro and in vivo meclonazepam treatment. These data confirm that meclonazepam causes extensive tegument damage and directly kills parasites, as measured by pro-apoptotic caspase activation. In vivo meclonazepam exposure results in differential expression of many genes that are divergent in parasitic flatworms, as well as several gene products implicated in blood feeding and regulation of hemostasis in other parasites. Many of these transcripts are also differentially expressed with praziquantel exposure, which may reflect a common schistosome response to the two drugs. However, despite these similarities in drug response, praziquantel-resistant parasites retain susceptibility to meclonazepam’s schistocidal effects. These data provide new insight into the mechanism of antischistosomal benzodiazepines, resolving similarities and differences with the current frontline therapy, praziquantel.