Biology subjects

Tham, W.-H.

Publications and source records attributed to Tham, W.-H..

At least 19 recordsLinked to original sources

bioRxiv2026

Nanobodies against Plasmodium vivax AMA1 block parasite invasion

Plasmodium vivax is the most widespread malaria species to infect human populations. P. vivax invades young red cells called reticulocytes to replicate and proliferate. An essential protein in parasite invasion is the Apical Membrane Antigen 1, PvAMA1, which forms the tight junction between parasite and red cell membrane through its interaction with Rhoptry Neck Protein 2, PvRON2. Here we report the identification of PvAMA1 nanobodies using phage display in combination with Sanger sequencing, next-generation sequencing and structure prediction tools. We show that these nanobodies bind with high affinities and across several different epitopes on PvAMA1. Several nanobodies recognize PvAMA1 in merozoites of late-stage schizonts and inhibit parasite invasion of red blood cells. These growth inhibitory nanobodies bind to conformational epitopes of PvAMA1 and most bind to a site distinct from a known potent human monoclonal antibody 826827. Crystal structures of two inhibitory nanobodies in complex with PvAMA1 show that they bind novel neutralizing epitopes which are outside the hydrophobic groove that contacts PvRON2. Furthermore, we show that two other PvAMA1 nanobodies also inhibit Plasmodium falciparum invasion. These results demonstrate the potential of PvAMA1 nanobodies to inhibit malaria parasite invasion across multiple Plasmodium species.

microbiology↗

bioRxiv2026

Structural basis of coiled-coil domain-mediated inhibition of A. baumannii AbTir NAD+ nucleosidase activity

TIR (Toll/interleukin-1 receptor) domains are central components of innate immunity and cell-death pathways across diverse forms of life. In bacteria, TIR domain-containing proteins participate in diverse antiphage defence systems, several of which rely on NADase activity carried out by TIR domains. This nucleosidase activity is often regulated by additional domains that modulate activation and specificity in response to infection. AbTir, a TIR domain-containing protein from Acinetobacter baumannii, contains an N-terminal CC domain that negatively regulates its NADase activity, but the structural basis of this regulation remains unknown. Here, we used X-ray crystallography to investigate the molecular basis of CC domain-mediated regulation in AbTir. We determined the crystal structure of full-length AbTir in complex with a nanobody, revealing a symmetric AbTir TIR dimer in which only one of the two symmetry-related CC helices is accommodated at the dimer surface, breaking the overall symmetry of the full-length protein. Structural comparisons with other bacterial TIR domains suggest that such TIR dimers likely represent an inactive state, in which the NAD+-binding site has not yet formed. Phylogenetic analysis places AbTir within a family of bacterial TIR proteins associated with defence-related genomic loci, although phage-spot assays performed in the surrogate host Escherichia coli did not detect measurable antiphage activity. Together, these findings reveal an unusual structural mechanism of CC domain-mediated inhibition of a bacterial TIR NADase and expand our understanding of the mechanistic diversity of bacterial TIR proteins.

biochemistry↗

bioRxiv2026

Cryo-EM structure of human LRRC15 reveals the basis of therapeutic antibody recognition

Leucine-rich repeat containing 15 (LRRC15) is a structural uncharacterised membrane protein, which is highly upregulated in cancer-associated fibroblasts and enriched in desmoplastic tumours including pancreatic, breast and head-and-neck cancers, where it is associated with therapy resistance. The LRRC15-targeting antibody-drug conjugate samrotamab vedotin has advanced into Phase I clinical evaluation, yet mechanistic understanding of how samrotamab engages its target has remained elusive in the absence of structural data. Here, using hydrogen-deuterium exchange mass spectrometry and cryo-electron microscopy, we define the samrotamab epitope on LRRC15 and determine the structure of the LRRC15-samrotamab complex to 2.6 [A] resolution - the first high-resolution structure of this receptor. Samrotamab engages a membrane-proximal epitope within the C-terminal leucine-rich repeat region, leaving the canonical concave surface fully exposed for potential interactions. The lateral binding mode of samrotamab and the open arc geometry of LRRC15 provide a structural rationale for why antibody occupancy may leave signalling-competent surfaces intact. Guided by these insights, we computationally designed minibinders targeting the concave surface, identifying multiple binders with nanomolar affinity. Together, our work establishes the first structural framework for LRRC15, defines the molecular basis of therapeutic antibody recognition, and identifies the concave surface as a promising target for next-generation LRRC15-directed therapeutics.

biochemistry↗

bioRxiv2026

Nanobodies against Plasmodium adhesins that block receptor engagement and malaria parasite invasion

Malaria is caused by Plasmodium parasites, and its clinical symptoms are a result of parasite invasion of red blood cells and the subsequent cycles of replication and proliferation. In human populations, Plasmodium vivax is responsible for the most widely distributed recurring malaria infections whereas Plasmodium falciparum inflicts the most mortality and morbidity. One well-characterized family of adhesins involved in red blood cell invasion is the reticulocyte-binding-like protein homolog family, known as the RBL superfamily which includes the PfRh family in P. falciparum and PvRBP family in P. vivax. Here we report a collection of nanobodies against three members of this adhesin family, PfRh5, PfRh4 and PvRBP2b. Nanobodies against these Plasmodium adhesins bind with high affinity across several epitopes and can block receptor engagement and inhibit parasite invasion of red blood cells. Using computational design, we generated stabilized PfRh4 variants that encompass the conserved scaffold present in the PfRh and PvRBP families of adhesins and show that several variants with improved expression retained binding to mouse monoclonal antibodies, nanobodies and Complement Receptor 1, the human receptor for PfRh4. We also observed that most of the inhibitory nanobodies against the three antigens recognized the conserved structural scaffold that define this family of adhesins. These results demonstrate the potential of nanobodies to block malaria parasite invasion into red blood cells.

microbiology↗

bioRxiv2025

Crystal structure and nanobodies against domain 3 of the malaria parasite fusogen Plasmodium falciparum HAP2

Malaria parasites are transmitted to humans through a bite from an infected female Anopheles mosquito. Within the mosquito midgut, malaria parasite gametes are activated and undergo fertilisation. If parasite fertilisation is perturbed, this stops the transmission of malaria parasites from mosquito to human. One proposed target of transmission-blocking interventions is Plasmodium falciparum fusogen PfHAP2, which is essential for gamete fusion during parasite fertilisation. However, to date, no monoclonal antibodies or structures of PfHAP2 have been generated. We have identified nanobodies that bind specifically to domain 3 of PfHAP2 with nanomolar affinities, two of which show some cross-species reactivity with HAP2 of other Plasmodium species. The crystal structure of one nanobody in complex with domain 3 of PfHAP2 provides the first structural insights into this transmission-blocking target in P. falciparum.

microbiology↗

bioRxiv2025

Examining Selection Dynamics and Limitations in Multi-round Protein Selection of High Diversity Libraries

Proteins and peptides underpin essential biological functions and technological applications, from targeting disease-relevant interactions to providing broad enzymatic activities. However, engineering molecules with desired properties remains difficult, owing to complex sequence-structure-function relationships and the lack of data on specific systems. Experimental selection strategies, including directed evolution, phage display, and mRNA display, address this challenge by leveraging high diversity libraries and iterative enrichment under defined selection pressures. This allows for the identification of candidates without requiring extensive prior knowledge, and can generate extensive datasets for use in machine learning. While many selection systems exist, comparisons across different selection approaches are hindered by the lack of a unifying analytical framework. Here, we present a set of broadly applicable analyses for assessing selection dynamics in multi-round or multi-condition experiments, ranging from position level analysis of sequence properties to full sequence space mappings through protein language model embeddings. Using the toolset to analyze a variety of different datasets in parallel, we explore the potential effects of diversity, coverage, and reproducibility, offering generalizable insights to guide experimental design, interpretation, and troubleshooting across protein and peptide discovery platforms.

molecular biology↗

bioRxiv2025

alpseq: an open-source workflow to turbocharge nanobody discovery with high-throughput sequencing

Nanobodies have emerged as promising tools for many biotechnological applications due to their small size, high stability, and remarkable binding specificity. Next-Generation Sequencing (NGS) enables deep profiling of large nanobody libraries and panning campaigns, however the scale and diversity of nanobody NGS datasets presents a significant bioinformatic challenge. To this end, we have developed alpseq, an optimised, open-source software pipeline designed specifically for the efficient and accurate processing of NGS data from nanobody libraries and panning campaigns. alpseq is also paired with a PCR-free sequencing library preparation protocol to allow researchers to easily generate their own data while avoiding biases. The alpseq software pipeline is composed of two parts: a pre-processing module written in Nextflow efficiently handles raw nanobody reads in a single line of code. These results are then fed into the analysis module, which contains a comprehensive suite of functions for quality control, diversity analysis, identification of enriched sequences and clustering. alpseq also creates a user-friendly interactive report which empowers scientists to explore their data without the need for extensive bioinformatic experience. Sophisticated panning campaign designs are supported, such as replicates and comparisons between different pans to find cross-binding leads. alpseq thus generates insights into the nanobody selection process and delivers a list of lead candidates for further experimental validation and downstream applications. alspeq is available at https://github.com/kzeglinski/alpseq.

bioinformatics↗

bioRxiv2025

A novel chimeric coronavirus spike vaccine combining SARS-CoV-2 RBD and scaffold domains from HKU-1 elicits potent neutralising antibody responses

The receptor binding domain (RBD) of the SARS-CoV-2 spike is the major target for neutralising antibodies elicited by current vaccines. Using small domains such as the RBD as vaccine immunogens, however, may constrain the availability of CD4 T follicular helper (TFH) cells and impact immunogenicity. We engineered a novel chimeric trimeric RBD (CTR) glycoprotein, replacing the RBD of human coronavirus HKU-1 spike with SARS-CoV-2 RBD of either ancestral (WT) or Omicron BA.2 strains. This strategy maintains a native trimeric conformation of the RBD, while providing additional sources of CD4 T cell help via the HKU-1 spike scaffold. In C57BL/6 mice, CTR-BA.2 prime-boost vaccination elicited high anti-BA.2-RBD IgG and neutralising titres, matching responses in animals immunised with native SARS-CoV-2 spike proteins. GC B cells elicited by CTR-BA.2 were predominantly WT+/BA.2+ cross- reactive, and TFH cells predominantly recognised HKU-1 epitopes, demonstrating scaffold-directed T cell help. Macaques prime-boost immunised with CTR-WT similarly elicited high anti-RBD IgG, anti-spike IgG and neutralising responses, comparable to native spike-vaccinated animals. In draining lymph nodes of CTR-WT vaccinated macaques, RBD-specific GC B cells were present at elevated levels. In contrast to the murine studies, lymph node-draining TFH responses in macaques were broadly elicited against RBD, NTD/S2 or HKU-1-derived peptides. Although native SARS- CoV-2 spike was also highly immunogenic in animal models, our findings establish the chimeric glycoprotein design as a strategy to overcome the poor immunogenicity of the SARS-CoV-2 RBD by engaging CD4 TFH cells, while maintaining the ability to elicit protective neutralising responses. One sentence summaryA chimeric glycoprotein design preserves SARS-CoV-2 RBD antigenic conformation enabling elicitation of neutralising responses, while allowing recruitment of HKU-1 scaffold-directed CD4 helper responses to support the humoral response.

immunology↗

bioRxiv2025

Navigating parasite antigen genetic diversity in the design of Plasmodium vivax serological exposure markers for malaria

BackgroundPlasmodium vivax poses a major obstacle to malaria elimination because it can lie dormant in the liver for weeks or months before reactivating and causing a relapse of infection. These dormant forms (hypnozoites) cannot be detected using standard diagnostics, but recent P. vivax exposure and by proxy, hypnozoite carriage, can be inferred using antibody-based tests (serological markers). In this study, we examined how genetic variation in P. vivax affects the utility of these antibody markers, and whether redesigned antigens could improve performance. MethodsWe analysed global P. vivax genetic data to assess variation in leading serological markers. Based on this, we produced new antigen versions (haplotypes) that better reflect global sequence diversity, compared to the commonly used reference strain (Sal-1). Antibody responses against these new constructs were then tested using samples from well-characterised cohorts in Brazil and Thailand. Antibody levels were assessed in relation to how recently participants had a qPCR-detectable blood-stage P. vivax infection. We compared the ability of the haplotypes and reference constructs to correctly identify individuals infected within the prior 9-months. FindingsExtensive genetic diversity was identified in two P. vivax antigens, DBPII and MSP5. Several antigens had large numbers of circulating haplotypes globally, with the percentage with similar sequence identity to the reference Sal-1 ranging from 0.4% (MSP5) to 99% (S16). Two antigens exhibited strong differences in immunogenicity by region and construct (RBP2a and DBPII). However, for most proteins (5 out of 8), these differences had little impact on the accuracy of identifying recent exposure. In cases where performance was affected (e.g. RBP2a), this could be overcome by adding multiple antigens into the classification model. InterpretationEven highly diverse antigens can be effective serological exposure markers. Our findings highlight the importance of testing the impact of genetic diversity when designing serological tests and suggest practical strategies, such as using a mix of antigens, to ensure consistent performance across regions.

microbiology↗

bioRxiv2025

Host iron deficiency protects against Plasmodium infection and drives parasite molecular reprofiling.

Iron deficiency, anemia and Plasmodium infection represent significant global health challenges with overlapping geographical distributions, particularly affecting pregnant women in Africa, yet the mechanisms underlying their interactions remain poorly understood. We employed a multilayered approach combining clinical data from Malawian pregnant women (n=711) in the REVAMP trial, a genetic mouse model (Tmprss6-knockout), and in vitro P. falciparum cultures to clarify associations between iron status and malaria susceptibility. Iron deficiency was associated with 50% reduced P. falciparum parasitemia in pregnant women (95% CI [30%-64%], p<0.0001), while iron-deficient mice exhibited significantly improved survival against P. berghei (median 15.5 days vs. 7.0 days for WT mice) and protection from cerebral malaria (83% vs 17% survival). Iron chelation induced substantial transcriptomic and proteomic changes in cultured parasites, affecting host cell invasion and nutrient acquisition processes. Importantly, intravenous iron supplementation did not increase subsequent parasitemia when coupled with malaria prevention. These findings demonstrate that iron deficiency protects against Plasmodium infection and support WHO recommendations for iron supplementation in malaria-endemic regions when combined with adequate malaria prevention strategies.

cell biology↗

bioRxiv2025

Pfs48/45 nanobodies block Plasmodium falciparum transmission

Malaria parasite fertilisation occurs within the Anopheles mosquito midgut. Interventions that inhibit parasite fertilisation prevent ongoing transmission and are important for malaria elimination efforts. Pfs48/45 and Pfs230 are two leading transmission-blocking vaccine candidates. Both proteins form a complex on the surface of sexual stage parasites and are essential for male fertility. Here we have identified nanobodies against Pfs48/45 that recognise gametocytes and have strong transmission-reducing activity. The crystal structure of our most potent nanobody in complex with Pfs48/45 reveals it binds a distinct epitope to TB31F, a leading transmission-blocking monoclonal antibody. In addition, we generated bispecific nanobodies that can target both Pfs48/45 and Pfs230 simultaneously and are fused to a human Fc domain. Our results show that these bispecific nanobodies recognise both Pfs48/45 and Pfs230 and reduce malaria parasite fertilisation in Anopheles stephensi. These results demonstrate the potential of nanobodies as a versatile antibody format that can reduce malaria transmission. Author SummaryMalaria is spread when an infected Anopheles mosquito bites a human. Within the female Anopheles mosquito, malaria parasite fertilisation occurs in the mosquito midgut. If you inhibit parasite fertilisation in the mosquito, you can prevent onward transmission of the malaria parasite from mosquito to humans. Transmission blocking vaccines work by stopping parasite fertilisation and development in the mosquito and are key for malaria elimination by preventing community spread. Pfs48/45 and Pfs230 are two leading transmission-blocking vaccine candidates, and both are critical for male fertility. Here we describe the generation of nanobodies that target Pfs48/45. We show that when nanobodies against Pfs48/45 were added to infected blood meals for Anopheles mosquitoes, the nanobodies significantly reduced parasite transmission. In addition, we generated bispecific nanobodies that target both Pfs48/45 and Pfs230 and these bispecific nanobodies also significantly reduced oocyst development. Our work demonstrates the potential of nanobodies as a versatile antibody format that can reduce malaria transmission.

microbiology↗

bioRxiv2025

Cryo-EM structure of endogenous Plasmodium falciparum Pfs230 and Pfs48/45 fertilization complex

Plasmodium falciparum Pfs230 and Pfs48/45, part of a core fertilization complex, are leading malaria transmission-blocking vaccine candidates. However, how the two proteins interact is unknown. Here we report a 3.36 [A] resolution cryo-electron microscopy structure of the endogenous Pfs230-Pfs48/45 complex. We show that Pfs48/45 interacts with Pfs230 domains 13 and 14, domains that are not included in current Pfs230 vaccine immunogens. Using a transgenic parasite line with a domain 13 to 14 deletion, we show that these domains are essential for Pfs230 localization on the gamete surface. Furthermore, this line significantly reduced oocyst formation in the mosquito midgut, showing that the presence of Pfs230 domains 13 and 14 is critical for successful fertilization. Nanobodies against domains 13 and 14 inhibit Pfs230-Pfs48/45 complex formation, reduce transmission and structural analyses reveal their binding epitopes. Furthermore, domains 13 and 14 are targets of naturally acquired immunity and when delivered as mRNA-LNP immunizations induce potent immune responses and blocked transmission of malaria parasites. Our comprehensive structural insights on a core P. falciparum fertilization complex will guide the design of novel transmission-blocking vaccine candidates against malaria.

microbiology↗

bioRxiv2024

Cavin1 binding nanobodies reveal structural flexibility and regulated interactions of the N-terminal coiled-coil domain

Caveolae are abundant plasma membrane structures that regulate signalling, membrane homeostasis, and mechanoprotection. Their formation is driven by caveolins and cavins and their coordinated interactions with lipids. We have developed nanobodies against the trimeric HR1 coiled-coil domain of Cavin1. We identify specific nanobodies that do not perturb Cavin1 membrane binding and localise to caveolae when expressed in cells. The crystal structure of a nanobody HR1 complex reveals a symmetric 3:3 architecture as validated by mutagenesis. In this structure, the C-terminal half of the HR1 domain is disordered suggesting the nanobody has stabilised an open conformation previously identified as important for membrane interactions. A phosphomimic mutation in a Thr-Ser pair proximal to this region reveals selective regulation of Cavin2/Cavin3 association. These studies provide new insights into Cavin domains required for assembly of multiprotein caveolar assemblies and describe new nanobody tools for structural and functional studies of caveolae. SUMMARY STATEMENTNanobodies are reported that can label the Cavin1 protein and caveola structures in cells.

cell biology↗

bioRxiv2024

Potent AMA1-specific human monoclonal antibody against P. vivax Pre-erythrocytic and Blood Stages

New therapeutics are necessary for preventing Plasmodium vivax malaria due to easy transmissibility and dormancy in the liver that increases the clinical burden due to recurrent relapse. We isolated 12 Pv Apical Membrane Antigen 1 (PvAMA1) specific human monoclonal antibodies from Peripheral Blood Mononuclear Cells of a Pv-exposed individual. PvAMA1 is essential for sporozoite and merozoite invasion, making it a unique therapeutic target. HumAb 826827 blocked the invasion of human erythrocytes using Pv clinical isolates and inhibited sporozoite invasion of human hepatocytes in vitro (IC50 of 0.3 - 3.7 {micro}g/mL). It also significantly reduced liver infection of chimeric FRG-humHep mice in vivo. The crystal structure of rPvAMA1 bound to 826827 shows that 826827 partially occupies the highly conserved hydrophobic groove in PvAMA1 that binds its known receptor, RON2. We have isolated a potent humAb that is isolate-transcendent, blocks both pre-erythrocytic and blood stage infection, and could be a new therapy for Pv.

immunology↗

bioRxiv2023

Aryl amino acetamides prevent the development of Plasmodium falciparum rings via inhibition of the lipid transfer protein PfSTART1

With resistance to most antimalarials increasing, it is imperative that new antimalarial drugs are developed to replace or complement front-line artemisinin therapies. We previously identified an aryl acetamide compound, MMV006833 (M-833), that inhibited ring development of newly invaded merozoites. Here, we selected parasites resistant to M-833 and identified independent mutations arising in the START lipid transfer protein (PF3D7_0104200, PfSTART1). Introduction of the identified PfSTART1 mutations into wildtype parasites reproduced resistance to both M-833 and highly potent analogues, confirming PfSTART1 mutations were sufficient to confer resistance. The analogues bound to recombinant PfSTART1 with nanomolar affinity. We also demonstrated selective PfSTART1 engagement by the analogues using organic solvent-based Proteome Integral Solubility Alteration (Solvent PISA) assay for the first time in Plasmodium. Imaging of newly invaded merozoites showed the inhibitors prevented the conversion into larger amoeboid ring-stage parasites potentially through the inhibition of phospholipid transfer from the parasite to the encasing parasitophorous vacuole membrane (PVM) and/or within the parasite. We show that these PfSTART1 inhibitors also block transmission. With multiple stages of the parasites lifecycle being targeted by PfSTART1 inhibitors, this protein therefore represents a novel drug target with a new mechanism of action.

cell biology↗

bioRxiv2022

Parallel use of pluripotent human stem cell lung and heart models provide new insights for treatment of SARS-CoV-2

SARS-CoV-2 primarily infects the respiratory tract, but pulmonary and cardiac complications occur in severe COVID-19. To elucidate molecular mechanisms in the lung and heart, we conducted paired experiments in human stem cell-derived lung alveolar type II (AT2) epithelial cell and cardiac cultures infected with SARS-CoV-2. With CRISPR- Cas9 mediated knock-out of ACE2, we demonstrated that angiotensin converting enzyme 2 (ACE2) was essential for SARS-CoV-2 infection of both cell types but further processing in lung cells required TMPRSS2 while cardiac cells required the endosomal pathway. Host responses were significantly different; transcriptome profiling and phosphoproteomics responses depended strongly on the cell type. We identified several antiviral compounds with distinct antiviral and toxicity profiles in lung AT2 and cardiac cells, highlighting the importance of using several relevant cell types for evaluation of antiviral drugs. Our data provide new insights into rational drug combinations for effective treatment of a virus that affects multiple organ systems. One-sentence summaryRational treatment strategies for SARS-CoV-2 derived from human PSC models

microbiology↗

bioRxiv2021

An iTSC-derived placental model of SARS-CoV-2 infection unveils ACE2-dependent susceptibility in syncytiotrophoblasts

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causing coronavirus disease 2019 (COVID-19) has caused a global health crisis. The primary site of infection is in the respiratory tract but the virus has been associated with a variety of complications involving the gastrointestinal and cardiovascular systems. Since the virus affects a variety of tissue types, there has been interest in understanding SARS-CoV-2 infection in early development and the placenta. ACE2 and TMPRSS2, two genes that are critical for SARS-CoV-2 virus entry are expressed in placenta-specific cell types including extravillous trophoblasts (EVTs) and especially, syncytiotrophoblasts (STs). The potential of SARS-CoV-2 to infect these placental cells and its effect on placental development and function is still unclear. Furthermore, it is crucial to understand the possible mechanism of vertical transmission of SARS-CoV-2 through the placenta. Here, we developed an in vitro model of SARS-CoV-2 infection of placental cell types using induced trophoblast stem cells (iTSCs). This model allowed us to show that STs but not EVTs are infected. Importantly, infected STs lack the expression of key differentiation genes, lack typically observed differentiated morphology and produce significantly lower human chorionic gonadotropin (HCG) compared to non-infected controls. We also show that an anti-ACE2 antibody prevents SARS-CoV-2 infection and restores normal ST differentiation and function. We highlight the establishment of a platform to study SARS-CoV-2 infection in early placental cell types, which will facilitate investigation of antiviral therapy to protect the placenta during early pregnancy and development.

cell biology↗

bioRxiv2021

Naturally acquired antibody kinetics against Plasmodium vivax antigens in people from a low malaria transmission region in western Thailand

Plasmodium vivax is the dominant Plasmodium spp. causing the disease malaria in low-transmission regions outside of Africa. These regions often feature high proportions of asymptomatic patients with sub-microscopic parasitaemia and relapses. Naturally acquired antibody responses are induced after Plasmodium infection, providing partial protection against high parasitaemia and clinical episodes. However, previous work has failed to address the presence and maintenance of such antibody responses to P. vivax particularly in low-transmission regions. We followed 34 patients in western Thailand after symptomatic P. vivax infections to monitor antibody kinetics over 9 months, during which no recurrent infections occurred. We assessed total IgG, IgG subclass and IgM levels to up to 52 P. vivax proteins every 2-4 weeks using a multiplexed Luminex(R) assay, and identified protein-specific variation in antibody longevity. Generally, an increase in antibody level was observed within 1-week post symptomatic infection, followed by an exponential decay of different rates. We observed mostly IgG1 dominance and IgG3 sub-dominance in this population. IgM responses followed similar kinetic patterns to IgG, with some proteins unexpectedly inducing long-lived IgM responses. We also monitored antibody responses against 27 IgG-immunogenic antigens in 30 asymptomatic individuals from a similar region. Our results demonstrate that most antigens induced robust and long-lived total IgG responses following asymptomatic infections in the absence of (detected) boosting infections. Our work provides new insights into the development and maintenance of naturally acquired immunity to P. vivax and will guide the potential use of serology to indicate immune status and/or identify populations at risk.

microbiology↗