Plasmodium vivax is the main cause of malaria outside of sub-Saharan Africa, and in many settings it presents significant challenges to malaria elimination efforts. Despite some control successes in the Americas, regional annual case counts of malaria have increased by over 25% between 2014 and 2023, largely driven by P. vivax. Genomic surveillance can play a key role in understanding the extent to which disease persistence represents indigenous transmission as opposed to introduction of new strains through migration, and whether specific variants evade control measures. Efforts to make P. vivax genomic surveillance more cost-effective have led to the development of targeted sequencing-based methods, which strike a varying balance between assay sensitivity and breadth/informativeness. We introduce two new highly sensitive multiplexed amplicon sequencing panels for P. vivax: PvGTSeq and PvCRiSP. PvGTSeq requires selective whole-genome amplification (sWGA) and contains 249 amplicons-36 for antimalarial resistance and 213 for population structure-optimized for Latin America but applicable to all continents. PvCRiSP features four highly polymorphic amplicons that operate without sWGA and is designed to estimate complexity of infection (COI), identify instances of clonal transmission, and characterize recurrent episodes. Both panels use a single multiplex PCR with non-proprietary reagents, achieve ≥75% amplicon recovery at parasitemias as low as five parasites/μL, and PvCRiSP remains effective with low quality DNA. PvGTSeq showed high sequencing accuracy (error rate 3.85e-4% - 2.87e-3%), and both panels efficiently detected alleles from minority clones in simulated polyclonal infections. We validated both panels with samples from Colombia, Guyana, Honduras, Panama, and Venezuela, and performed in-silico assessments using data from 16 countries worldwide, confirming that these two panels have high power to discriminate samples and assign global geographic origin to imported cases. These panels will therefore be useful tools for P. vivax molecular surveillance in diverse geographic settings.
Vector control remains an important strategy worldwide to prevent human infection with pathogens transmitted by arthropods. Vector control strategies rely on accurate identification of vector taxa along with vector-specific biological indicators such as feeding ecology, infection prevalence and insecticide resistance. Multiple 'DNA barcoding' protocols have been published over the past several decades to support these applications, generally relying on informal manual approaches such as BLAST to assign taxonomic identity to the resulting sequences. We present a standardised informatic pipeline for analysis of DNA barcoding data from dipteran vectors, VecTreeID, that uses short-read amplicon sequencing (AmpSeq) coupled with sequence similarity assessment (BLAST) and an evolutionary placement algorithm (EPA-ng) to achieve vector taxonomic identification, capture bionomic features (blood and plant meal sources), determine Plasmodium infection status (for anopheline mosquitoes) and detect target-site insecticide resistance mutations. The VecTreeID pipeline provides uncertainty in assignment through identifications at varying levels of taxonomic rank, a feature missing from many approaches to DNA barcoding, but important given gaps and labelling problems in public sequence databases. We validated an Illumina-based implementation of VecTreeID on laboratory and field samples, and find that the blood meal amplicons can detect vertebrate DNA sequences up to 36 h post-feeding, and that short-read sequencing data are capable of sensitively detecting minor sequences in DNA mixtures representing multi-species blood or nectar meals. This high-throughput VecTreeID approach empowers researchers and public health professionals to survey and control arthropod disease vectors consistently and effectively.
10046 Background: Preclinical work demonstrated antitumor activity of VCN-01 (oncolytic adenovirus targeting the E2F pathway and expressing hyaluronidase) for retinoblastoma. We report this first-in-children study aiming to assess its safety and efficacy. Methods: Patients with intraocular retinoblastoma who failed conservative therapy facing imminent enucleation were eligible for this phase I, dose-escalation study (NCT03284268) with two dose levels of VCN-01 intravitreal injection (2E+9 vp/eye per dose for the first patient) and 2E+10 vp/eye dose in two doses every two weeks for the remaining 8. Dose limiting toxicity (DLT) was defined as ≥ grade IV ocular toxicity or ≥ grade III systemic toxicity according to CTCAEv04. Response assessed by RB-RECIST criteria and toxicity were evaluated at day 42 of the first injection. Results: Thirteen patients (4 screening failures) were enrolled. Out of the 9 treated patients, five had bilateral retinoblastoma. There was no DLT. 7/9 patients experienced adverse reactions. being uveitis the most common (7/9 patients, G3 in four). From the second patient onwards, all patients received pre-emptive oral and/or topical steroids to prevent uveitis. Uveitis was improved or resolved at day 42 in 7 patients. One patient with G3 uveitis did not receive the second dose because of medical decision and also experienced glaucoma requiring treatment. No systemic toxicities occurred. VCN-01 caused reversible changes in electroretinograms due to turbidity. Viral particles were not found in the healthy retina in enucleated eyes. No VCN-01 genomes in peripheral blood were detected in any case. At 42 days of the first injection, 5 patients achieved partial response, 3 stable disease and one progressive disease. Subsequent eye-conservative treatment was administered to 5 patients and 3 eyes are preserved with vision (follow-up 12-49 months). The remaining 6 eyes were enucleated because of refractory tumor. No extraocular relapse occurred. Conclusions: VCN01 was safe, being uveitis the most common adverse effect. VCN-01 did not cause retinal toxicity. The response in these heavily pre-treated eyes was encouraging. Clinical trial information: NCT03284268 .
BackgroundPs48/45, a Plasmodium gametocyte surface protein, is a promising candidate for malaria transmission-blocking (TB) vaccine. Due to its relevance for a multispecies vaccine, we explored the cross-reactivity and TB activity of a recombinant P. vivax Ps48/45 protein (rPvs48/45) with plasma from P. falciparum-exposed African donors.MethodsrPvs48/45 was produced in Chinese hamster ovary cell lines and tested by ELISA for cross-reactivity with plasma from Burkina Faso, Tanzania, Mali, and Nigeria. In addition, BALB/c mice were immunized with the rPvs48/45 protein formulated in Montanide ISA-51 and inoculated with a crude extract of P. falciparum NF-54 gametocytes to evaluate the parasite-boosting effect on rPvs48/45 antibody titers. Specific anti-rPvs48/45 IgG purified from African plasma was used to evaluate the ex vivo TB activity on P. falciparum, using standard mosquito membrane feeding assays (SMFA).ResultsrPvs48/45 protein showed cross-reactivity with plasma of individuals from all four African countries, in proportions ranging from 94% (Tanzania) to 40% (Nigeria). Also, the level of cross-reactive antibodies varied significantly between countries (p < 0.0001), with a higher antibody level in Mali and the lowest in Nigeria. In addition, antibody levels were higher in adults ( ≥ 17 years) than young children ( ≤ 5 years) in both Mali and Tanzania, with a higher proportion of responders in adults (90%) than in children (61%) (p < 0.0001) in Mali, where male (75%) and female (80%) displayed similar antibody responses. Furthermore, immunization of mice with P. falciparum gametocytes boosted anti-Pvs48/45 antibody responses, recognizing P. falciparum gametocytes in indirect immunofluorescence antibody test. Notably, rPvs48/45 affinity-purified African IgG exhibited a TB activity of 61% against P. falciparum in SMFA.ConclusionPlasma from African volunteers predominantly exposed to P. falciparum cross-recognized the rPvs48/45 protein. This, together with the functional activity of IgG, warrants further studies for the potential development of a P. vivax and P. falciparum cross-protective TB vaccine.
Malaria sterile immunity has been reproducibly induced by immunization with Plasmodium radiation-attenuated sporozoites (RAS). Analyses of sera from RAS-immunized individuals allowed the identification of P. falciparum antigens, such as the circumsporozoite protein (CSP), the basis for the RTS, S and R21Matrix-M vaccines. Similar advances in P. vivax (Pv) vaccination have been elusive. We previously reported 42% (5/12) of sterile protection in malaria-unexposed, Duffy-positive (Fy +) volunteers immunized with PvRAS followed by a controlled human malaria infection (CHMI). Using a custom protein microarray displaying 515 Pv antigens, we found a significantly higher reactivity to PvCSP and one hypothetical protein (PVX_089630) in volunteers protected against P. vivax infection. In mock-vaccinated Fy + volunteers, a strong antibody response to CHMI was also observed. Although the Fy- volunteers immunized with non-irradiated Pv-infected mosquitoes (live sporozoites) did not develop malaria after CHMI, they recognized a high number of antigens, indicating the temporary presence of asexual parasites in peripheral blood. Together, our findings contribute to the understanding of the antibody response to P. vivax infection and allow the identification of novel parasite antigens as vaccine candidates. Trial registration: ClinicalTrials.gov number: NCT 01082341.
