Phenotypic changes of γδ T cells in Plasmodium falciparum placental malaria and pregnancy outcomes in women at delivery in Cameroon.

Introduction: Depending on the microenvironment, γδ T cells may assume characteristics similar to those of Th1, Th2, Th17, regulatory T cells or antigen presenting cells. Despite the wide documentation of the effect of Th1/Th2 balance on pregnancy associated malaria and outcomes, there are no reports on the relationship between γδ T cell phenotype change and Placental Malaria (PM) with pregnancy outcomes. This study sought to investigate the involvement of γδ T cells and its subsets in placental Plasmodium falciparum malaria. Methods: In a case-control study conducted in Yaoundé, Cameroon from March 2022 to May 2023, peripheral, placental and cord blood samples were collected from 50 women at delivery (29 PM negative: PM- and 21 PM positive: PM+; as diagnosed by light microscopy). Hemoglobin levels were measured using hemoglobinometer. PBMCs, IVBMCs and CBMCs were isolated using histopaque-1077 and used to characterize total γδ T cell populations and subsets (Vδ1+, Vδ2+, Vδ1-Vδ2-) by flow cytometry. Results: Placental Plasmodium falciparum infection was associated with significant increase in the frequency of total γδ T cells in IVBMC and of the Vδ1+ subset in PBMC and IVBMC, but decreased frequency of the Vδ2+ subset in PBMC and IVBMC. The expression of the activation marker: HLA-DR, and the exhaustion markers (PD1 and TIM3) within total γδ T cells and subsets were significantly up-regulated in PM+ compared to PM- group. The frequency of total γδ T cells in IVBMC, TIM-3 expression within total γδ T cells and subsets in IVBMC, as well as HLA-DR expression within total γδ T cells and Vδ2+ subset in IVBMC were negatively associated with maternal hemoglobin levels. Furthermore, the frequency of total γδ T cells in PBMC and PD1 expression within the Vδ2+ subset in CBMC were negatively associated with birth weight contrary to the frequency of Vδ1-Vδ2- subset in PBMC and HLA-DR expression within the Vδ2+ subset in IVBMC which positively associated with maternal hemoglobin level and birth weight, respectively. Conclusion: The data indicate up-regulation of activated and exhausted γδ T cells in Plasmodium falciparum placental malaria, with effects on pregnancy outcomes including maternal hemoglobin level and birth weight.

SOD3 suppresses early cellular immune responses to parasite infection.

Host immune responses are tightly controlled by various immune factors during infection, and protozoan parasites also manipulate the immune system to evade surveillance, leading to an evolutionary arms race in host‒pathogen interactions; however, the underlying mechanisms are not fully understood. We observed that the level of superoxide dismutase 3 (SOD3) was significantly elevated in both Plasmodium falciparum malaria patients and mice infected with four parasite species. SOD3-deficient mice had a substantially longer survival time and lower parasitemia than control mice after infection, whereas SOD3-overexpressing mice were much more vulnerable to parasite infection. We revealed that SOD3, secreted from activated neutrophils, bound to T cells, suppressed the interleukin-2 expression and concomitant interferon-gamma responses crucial for parasite clearance. Overall, our findings expose active fronts in the arms race between the parasites and host immune system and provide insights into the roles of SOD3 in shaping host innate immune responses to parasite infection.

Development of an improved blood-stage malaria vaccine targeting the essential RH5-CyRPA-RIPR invasion complex.

Reticulocyte-binding protein homologue 5 (RH5), a leading blood-stage Plasmodium falciparum malaria vaccine target, interacts with cysteine-rich protective antigen (CyRPA) and RH5-interacting protein (RIPR) to form an essential heterotrimeric "RCR-complex". We investigate whether RCR-complex vaccination can improve upon RH5 alone. Using monoclonal antibodies (mAbs) we show that parasite growth-inhibitory epitopes on each antigen are surface-exposed on the RCR-complex and that mAb pairs targeting different antigens can function additively or synergistically. However, immunisation of female rats with the RCR-complex fails to outperform RH5 alone due to immuno-dominance of RIPR coupled with inferior potency of anti-RIPR polyclonal IgG. We identify that all growth-inhibitory antibody epitopes of RIPR cluster within the C-terminal EGF-like domains and that a fusion of these domains to CyRPA, called "R78C", combined with RH5, improves the level of in vitro parasite growth inhibition compared to RH5 alone. These preclinical data justify the advancement of the RH5.1 + R78C/Matrix-M™ vaccine candidate to Phase 1 clinical trial.

Immunization with PfGBP130 generates antibodies that inhibit RBC invasion by P. falciparum parasites.

Background: Despite decades of effort, Plasmodium falciparum malaria remains a leading killer of children. The absence of a highly effective vaccine and the emergence of parasites resistant to both diagnosis as well as treatment hamper effective public health interventions. Methods and results: To discover new vaccine candidates, we used our whole proteome differential screening method and identified PfGBP130 as a parasite protein uniquely recognized by antibodies from children who had developed resistance to P. falciparum infection but not from those who remained susceptible. We formulated PfGBP130 as lipid encapsulated mRNA, DNA plasmid, and recombinant protein-based immunogens and evaluated the efficacy of murine polyclonal anti-PfGBP130 antisera to inhibit parasite growth in vitro. Immunization of mice with PfGBP130-A (aa 111-374), the region identified in our differential screen, formulated as a DNA plasmid or lipid encapsulated mRNA, but not as a recombinant protein, induced antibodies that inhibited RBC invasion in vitro. mRNA encoding the full ectodomain of PfGBP130 (aa 89-824) also generated parasite growth-inhibitory antibodies. Conclusion: We are currently advancing PfGBP130-A formulated as a lipid-encapsulated mRNA for efficacy evaluation in non-human primates.

Machine learning prediction of malaria vaccine efficacy based on antibody profiles.

Immunization through repeated direct venous inoculation of Plasmodium falciparum (Pf) sporozoites (PfSPZ) under chloroquine chemoprophylaxis, using the PfSPZ Chemoprophylaxis Vaccine (PfSPZ-CVac), induces high-level protection against controlled human malaria infection (CHMI). Humoral and cellular immunity contribute to vaccine efficacy but only limited information about the implicated Pf-specific antigens is available. Here, we examined Pf-specific antibody profiles, measured by protein arrays representing the full Pf proteome, of 40 placebo- and PfSPZ-immunized malaria-naïve volunteers from an earlier published PfSPZ-CVac dose-escalation trial. For this purpose, we both utilized and adapted supervised machine learning methods to identify predictive antibody profiles at two different time points: after immunization and before CHMI. We developed an adapted multitask support vector machine (SVM) approach and compared it to standard methods, i.e. single-task SVM, regularized logistic regression and random forests. Our results show, that the multitask SVM approach improved the classification performance to discriminate the protection status based on the underlying antibody-profiles while combining time- and dose-dependent data in the prediction model. Additionally, we developed the new fEature diStance exPlainabilitY (ESPY) method to quantify the impact of single antigens on the non-linear multitask SVM model and make it more interpretable. In conclusion, our multitask SVM model outperforms the studied standard approaches in regard of classification performance. Moreover, with our new explanation method ESPY, we were able to interpret the impact of Pf-specific antigen antibody responses that predict sterile protective immunity against CHMI after immunization. The identified Pf-specific antigens may contribute to a better understanding of immunity against human malaria and may foster vaccine development.

Repeat controlled human Plasmodium falciparum infections delay bloodstream patency and reduce symptoms.

Resistance to clinical malaria takes years to develop even in hyperendemic regions and sterilizing immunity has rarely been observed. To evaluate the maturation of the host response against controlled repeat exposures to P. falciparum (Pf) NF54 strain-infected mosquitoes, we systematically monitored malaria-naïve participants through an initial exposure to uninfected mosquitoes and 4 subsequent homologous exposures to Pf-infected mosquitoes over 21 months (n = 8 males) (ClinicalTrials.gov# NCT03014258). The primary outcome was to determine whether protective immunity against parasite infection develops following repeat CHMI and the secondary outcomes were to track the clinical signs and symptoms of malaria and anti-Pf antibody development following repeat CHMI. After two exposures, time to blood stage patency increases significantly and the number of reported symptoms decreases indicating the development of clinical tolerance. The time to patency correlates positively with both anti-Pf circumsporozoite protein (CSP) IgG and CD8 + CD69+ effector memory T cell levels consistent with partial pre-erythrocytic immunity. IFNγ levels decrease significantly during the participants' second exposure to high blood stage parasitemia and could contribute to the decrease in symptoms. In contrast, CD4-CD8 + T cells expressing CXCR5 and the inhibitory receptor, PD-1, increase significantly after subsequent Pf exposures, possibly dampening the memory response and interfering with the generation of robust sterilizing immunity.

Plasmodium falciparum AMA1 and CSP antigen diversity in parasite isolates from southern Ghana.

Introduction: Diversity in malarial antigens is an immune evasion mechanism that gives malaria parasites an edge over the host. Immune responses against one variant of a polymorphic antigen are usually not fully effective against other variants due to altered epitopes. This study aimed to evaluate diversity in the Plasmodium falciparum antigens apical membrane antigen 1 (PfAMA1) and circumsporozoite protein (PfCSP) from circulating parasites in a malaria-endemic community in southern Ghana and to determine the effects of polymorphisms on antibody response specificity. Methods: The study involved 300 subjects, whose P. falciparum infection status was determined by microscopy and PCR. Diversity within the two antigens was evaluated by msp2 gene typing and molecular gene sequencing, while the host plasma levels of antibodies against PfAMA1, PfCSP, and two synthetic 24mer peptides from the conserved central repeat region of PfCSP, were measured by ELISA. Results: Of the 300 subjects, 171 (57%) had P. falciparum infection, with 165 of the 171 (96.5%) being positive for either or both of the msp2 allelic families. Gene sequencing of DNA from 55 clonally infected samples identified a total of 56 non-synonymous single nucleotide polymorphisms (SNPs) for the Pfama1 gene and these resulted in 44 polymorphic positions, including two novel positions (363 and 365). Sequencing of the Pfcsp gene from 69 clonal DNA samples identified 50 non-synonymous SNPs that resulted in 42 polymorphic positions, with half (21) of these polymorphic positions being novel. Of the measured antibodies, only anti-PfCSP antibodies varied considerably between PCR parasite-positive and parasite-negative persons. Discussion: These data confirm the presence of a considerable amount of unique, previously unreported amino acid changes, especially within PfCSP. Drivers for this diversity in the Pfcsp gene do not immediately seem apparent, as immune pressure will be expected to drive a similar level of diversity in the Pfama1 gene.

Full-length MSP1 is a major target of protective immunity after controlled human malaria infection.

The merozoite surface protein 1 (MSP1) is the most abundant protein on the surface of the invasive merozoite stages of Plasmodium falciparum and has long been considered a key target of protective immunity. We used samples from a single controlled human malaria challenge study to test whether the full-length version of MSP1 (MSP1FL) induced antibodies that mediated Fc-IgG functional activity in five independent assays. We found that anti-MSP1FL antibodies induced complement fixation via C1q, monocyte-mediated phagocytosis, neutrophil respiratory burst, and natural killer cell degranulation as well as IFNγ production. Activity in each of these assays was strongly associated with protection. The breadth of MSP1-specific Fc-mediated effector functions was more strongly associated with protection than the individual measures and closely mirrored what we have previously reported using the same assays against merozoites. Our findings suggest that MSP1FL is an important target of functional antibodies that contribute to a protective immune response against malaria.

Immune responses associated with protection induced by chemoattenuated PfSPZ vaccine in malaria-naive Europeans.

Vaccination of malaria-naive volunteers with a high dose of Plasmodium falciparum sporozoites chemoattenuated by chloroquine (CQ) (PfSPZ-CVac [CQ]) has previously demonstrated full protection against controlled human malaria infection (CHMI). However, lower doses of PfSPZ-CVac [CQ] resulted in incomplete protection. This provides the opportunity to understand the immune mechanisms needed for better vaccine-induced protection by comparing individuals who were protected with those not protected. Using mass cytometry, we characterized immune cell composition and responses of malaria-naive European volunteers who received either lower doses of PfSPZ-CVac [CQ], resulting in 50% protection irrespective of the dose, or a placebo vaccination, with everyone becoming infected following CHMI. Clusters of CD4+ and γδ T cells associated with protection were identified, consistent with their known role in malaria immunity. Additionally, EMRA CD8+ T cells and CD56+CD8+ T cell clusters were associated with protection. In a cohort from a malaria-endemic area in Gabon, these CD8+ T cell clusters were also associated with parasitemia control in individuals with lifelong exposure to malaria. Upon stimulation with P. falciparum-infected erythrocytes, CD4+, γδ, and EMRA CD8+ T cells produced IFN-γ and/or TNF, indicating their ability to mediate responses that eliminate malaria parasites.

Breadth of Fc-mediated effector function correlates with clinical immunity following human malaria challenge.

Malaria is a life-threatening disease of global health importance, particularly in sub-Saharan Africa. The growth inhibition assay (GIA) is routinely used to evaluate, prioritize, and quantify the efficacy of malaria blood-stage vaccine candidates but does not reliably predict either naturally acquired or vaccine-induced protection. Controlled human malaria challenge studies in semi-immune volunteers provide an unparalleled opportunity to robustly identify mechanistic correlates of protection. We leveraged this platform to undertake a head-to-head comparison of seven functional antibody assays that are relevant to immunity against the erythrocytic merozoite stage of Plasmodium falciparum. Fc-mediated effector functions were strongly associated with protection from clinical symptoms of malaria and exponential parasite multiplication, while the gold standard GIA was not. The breadth of Fc-mediated effector function discriminated clinical immunity following the challenge. These findings present a shift in the understanding of the mechanisms that underpin immunity to malaria and have important implications for vaccine development.

IgG and IgM responses to the Plasmodium falciparum asexual stage antigens reflect respectively protection against malaria during pregnancy and infanthood.

BACKGROUND: Plasmodium falciparum malaria is a public health issue mostly seen in tropical countries. Until now, there is no effective malaria vaccine against antigens specific to the blood-stage of P. falciparum infection. Because the pathogenesis of malarial disease results from blood-stage infection, it is essential to identify the most promising blood-stage vaccine candidate antigens under natural exposure to malaria infection. METHODS: A cohort of 400 pregnant women and their infants was implemented in South Benin. An active and passive protocol of malaria surveillance was established during pregnancy and infancy to precisely ascertain malaria infections during the follow-up. Twenty-eight antibody (Ab) responses specific to seven malaria candidate vaccine antigens were repeatedly quantified during pregnancy (3 time points) and infancy (6 time points) in order to study the Ab kinetics and their protective role. Abs were quantified by ELISA and logistic, linear and cox-proportional hazard model were performed to analyse the associations between Ab responses and protection against malaria in mothers and infants, taking into account socio-economic factors and for infants an environmental risk of exposure. RESULTS: The levels of IgM against MSP1, MSP2 and MSP3 showed an early protective response against the onset of symptomatic malaria infections starting from the 18th month of life, whereas no association was found for IgG responses during infancy. In women, some IgG responses tend to be associated with a protection against malaria risk along pregnancy and at delivery, among them IgG3 against GLURP-R0 and IgG2 against MSP1. CONCLUSION: The main finding suggests that IgM should be considered in vaccine designs during infanthood. Investigation of the functional role played by IgM in malaria protection needs further attention.

Wolbachia infection-responsive immune genes suppress Plasmodium falciparum infection in Anopheles stephensi.

Wolbachia, a maternally transmitted symbiotic bacterium of insects, can suppress a variety of human pathogens in mosquitoes, including malaria-causing Plasmodium in the Anopheles vector. However, the mechanistic basis of Wolbachia-mediated Plasmodium suppression in mosquitoes is not well understood. In this study, we compared the midgut and carcass transcriptomes of stably infected Anopheles stephensi with Wolbachia wAlbB to uninfected mosquitoes in order to discover Wolbachia infection-responsive immune genes that may play a role in Wolbachia-mediated anti-Plasmodium activity. We show that wAlbB infection upregulates 10 putative immune genes and downregulates 14 in midguts, while it upregulates 31 putative immune genes and downregulates 15 in carcasses at 24 h after blood-fed feeding, the time at which the Plasmodium ookinetes are traversing the midgut tissue. Only a few of these regulated immune genes were also significantly differentially expressed between Wolbachia-infected and non-infected midguts and carcasses of sugar-fed mosquitoes. Silencing of the Wolbachia infection-responsive immune genes TEP 4, TEP 15, lysozyme C2, CLIPB2, CLIPB4, PGRP-LD and two novel genes (a peritrophin-44-like gene and a macro domain-encoding gene) resulted in a significantly greater permissiveness to P. falciparum infection. These results indicate that Wolbachia infection modulates mosquito immunity and other processes that are likely to decrease Anopheles permissiveness to Plasmodium infection.

A Pfs48/45-based vaccine to block Plasmodium falciparum transmission: phase 1, open-label, clinical trial.

BACKGROUND: The stalling global progress in malaria control highlights the need for novel tools for malaria elimination, including transmission-blocking vaccines. Transmission-blocking vaccines aim to induce human antibodies that block parasite development in the mosquito and mosquitoes becoming infectious. The Pfs48/45 protein is a leading Plasmodium falciparum transmission-blocking vaccine candidate. The R0.6C fusion protein, consisting of Pfs48/45 domain 3 (6C) and the N-terminal region of P. falciparum glutamate-rich protein (R0), has previously been produced in Lactococcus lactis and elicited functional antibodies in rodents. Here, we assess the safety and transmission-reducing efficacy of R0.6C adsorbed to aluminium hydroxide with and without Matrix-M™ adjuvant in humans. METHODS: In this first-in-human, open-label clinical trial, malaria-naïve adults, aged 18-55 years, were recruited at the Radboudumc in Nijmegen, the Netherlands. Participants received four intramuscular vaccinations on days 0, 28, 56 and 168 with either 30 µg or 100 µg of R0.6C and were randomised for the allocation of one of the two different adjuvant combinations: aluminium hydroxide alone, or aluminium hydroxide combined with Matrix-M1™ adjuvant. Adverse events were recorded from inclusion until 84 days after the fourth vaccination. Anti-R0.6C and anti-6C IgG titres were measured by enzyme-linked immunosorbent assay. Transmission-reducing activity of participants' serum and purified vaccine-specific immunoglobulin G was assessed by standard membrane feeding assays using laboratory-reared Anopheles stephensi mosquitoes and cultured P. falciparum gametocytes. RESULTS: Thirty-one participants completed four vaccinations and were included in the analysis. Administration of all doses was safe and well-tolerated, with one related grade 3 adverse event (transient fever) and no serious adverse events occurring. Anti-R0.6C and anti-6C IgG titres were similar between the 30 and 100 µg R0.6C arms, but higher in Matrix-M1™ arms. Neat participant sera did not induce significant transmission-reducing activity in mosquito feeding experiments, but concentrated vaccine-specific IgGs purified from sera collected two weeks after the fourth vaccination achieved up to 99% transmission-reducing activity. CONCLUSIONS: R0.6C/aluminium hydroxide with or without Matrix-M1™ is safe, immunogenic and induces functional Pfs48/45-specific transmission-blocking antibodies, albeit at insufficient serum concentrations to result in transmission reduction by neat serum. Future work should focus on identifying alternative vaccine formulations or regimens that enhance functional antibody responses. TRIAL REGISTRATION: The trial is registered with ClinicalTrials.gov under identifier NCT04862416.

Innate immune activation restricts priming and protective efficacy of the radiation-attenuated PfSPZ malaria vaccine.

A systems analysis was conducted to determine the potential molecular mechanisms underlying differential immunogenicity and protective efficacy results of a clinical trial of the radiation-attenuated whole-sporozoite PfSPZ vaccine in African infants. Innate immune activation and myeloid signatures at prevaccination baseline correlated with protection from P. falciparum parasitemia in placebo controls. These same signatures were associated with susceptibility to parasitemia among infants who received the highest and most protective PfSPZ vaccine dose. Machine learning identified spliceosome, proteosome, and resting DC signatures as prevaccination features predictive of protection after highest-dose PfSPZ vaccination, whereas baseline circumsporozoite protein-specific (CSP-specific) IgG predicted nonprotection. Prevaccination innate inflammatory and myeloid signatures were associated with higher sporozoite-specific IgG Ab response but undetectable PfSPZ-specific CD8+ T cell responses after vaccination. Consistent with these human data, innate stimulation in vivo conferred protection against infection by sporozoite injection in malaria-naive mice while diminishing the CD8+ T cell response to radiation-attenuated sporozoites. These data suggest a dichotomous role of innate stimulation for malaria protection and induction of protective immunity by whole-sporozoite malaria vaccines. The uncoupling of vaccine-induced protective immunity achieved by Abs from more protective CD8+ T cell responses suggests that PfSPZ vaccine efficacy in malaria-endemic settings may be constrained by opposing antigen presentation pathways.

The influence of neonatal BCG vaccination on in vitro cytokine responses to Plasmodium falciparum.

BACKGROUND: Bacillus Calmette-Guérin (BCG) vaccination has off-target protective effects against infections unrelated to tuberculosis. Among these, murine and human studies suggest that BCG vaccination may protect against malaria. We investigated whether BCG vaccination influences neonatal in vitro cytokine responses to Plasmodium falciparum. Blood samples were collected from 108 participants in the Melbourne Infant Study BCG for Allergy and Infection Reduction (MIS BAIR) randomised controlled trial (Clinical trials registration NCT01906853, registered July 2013), seven days after randomisation to neonatal BCG (n = 66) or no BCG vaccination (BCG-naïve, n = 42). In vitro cytokine responses were measured following stimulation with P. falciparum-infected erythrocytes (PfIE) or E. coli. RESULTS: No difference in the measured cytokines were observed between BCG-vaccinated and BCG-naïve neonates following stimulation with PfIE or E. coli. However, age at which blood was sampled was independently associated with altered cytokine responses to PfIE. Being male was also independently associated with increased TNF-a responses to both PfIE and E. coli. CONCLUSION: These findings do not support a role for BCG vaccination in influencing in vitro neonatal cytokine responses to P. falciparum. Older neonates are more likely to develop P. falciparum-induced IFN-γ and IFN-γ-inducible chemokine responses implicated in early protection against malaria and malaria pathogenesis.

Vaccines and monoclonal antibodies: new tools for malaria control.

SUMMARYMalaria remains one of the biggest health problems in the world. While significant reductions in malaria morbidity and mortality had been achieved from 2000 to 2015, the favorable trend has stalled, rather significant increases in malaria cases are seen in multiple areas. In 2022, there were 249 million estimated cases, and 608,000 malaria-related deaths, mostly in infants and children aged under 5 years, globally. Therefore, in addition to the expansion of existing anti-malarial control measures, it is critical to develop new tools, such as vaccines and monoclonal antibodies (mAbs), to fight malaria. In the last 2 years, the first and second malaria vaccines, both targeting Plasmodium falciparum circumsporozoite proteins (PfCSP), have been recommended by the World Health Organization to prevent P. falciparum malaria in children living in moderate to high transmission areas. While the approval of the two malaria vaccines is a considerable milestone in vaccine development, they have much room for improvement in efficacy and durability. In addition to the two approved vaccines, recent clinical trials with mAbs against PfCSP, blood-stage vaccines against P. falciparum or P. vivax, and transmission-blocking vaccine or mAb against P. falciparum have shown promising results. This review summarizes the development of the anti-PfCSP vaccines and mAbs, and recent topics in the blood- and transmission-blocking-stage vaccine candidates and mAbs. We further discuss issues of the current vaccines and the directions for the development of next-generation vaccines.

Bi-isotype immunoglobulins enhance antibody-mediated neutrophil activity against Plasmodium falciparum parasites.

Background: Malaria remains a major global health priority, and monoclonal antibodies (mAbs) are emerging as potential new tools to support efforts to control the disease. Recent data suggest that Fc-dependent mechanisms of immunity are important mediators of protection against the blood stages of the infection, but few studies have investigated this in the context of mAbs. We aimed to isolate mAbs agnostic to cognate antigens that target whole merozoites and simultaneously induce potent neutrophil activity measured by the level of reactive oxygen species (ROS) production using an antibody-dependent respiratory burst (ADRB) assay. Methods: We used samples from semi-immune adults living in coastal Kenya to isolate mAbs that induce merozoite-specific ADRB activity. We then tested whether modifying the expressed IgG1 isotype to an IgG-IgA Fc region chimera would enhance the level of ADRB activity. Results: We isolated a panel of nine mAbs with specificity to whole merozoites. mAb J31 induced ADRB activity in a dose-dependent fashion. Compared to IgG1, our modified antibody IgG-IgA bi-isotype induced higher ADRB activity across all concentrations tested. Further, we observed a negative hook effect at high IgG1 mAb concentrations (i.e., >200 µg/mL), but this was reversed by Fc modification. We identified MSP3.5 as the potential cognate target of mAb J31. Conclusions: We demonstrate an approach to engineer mAbs with enhanced ADRB potency against blood-stage parasites.

Positive-unlabeled learning identifies vaccine candidate antigens in the malaria parasite Plasmodium falciparum.

Malaria vaccine development is hampered by extensive antigenic variation and complex life stages of Plasmodium species. Vaccine development has focused on a small number of antigens, many of which were identified without utilizing systematic genome-level approaches. In this study, we implement a machine learning-based reverse vaccinology approach to predict potential new malaria vaccine candidate antigens. We assemble and analyze P. falciparum proteomic, structural, functional, immunological, genomic, and transcriptomic data, and use positive-unlabeled learning to predict potential antigens based on the properties of known antigens and remaining proteins. We prioritize candidate antigens based on model performance on reference antigens with different genetic diversity and quantify the protein properties that contribute most to identifying top candidates. Candidate antigens are characterized by gene essentiality, gene ontology, and gene expression in different life stages to inform future vaccine development. This approach provides a framework for identifying and prioritizing candidate vaccine antigens for a broad range of pathogens.

Expression of the MSPDBL2 antigen in a discrete subset of Plasmodium falciparum schizonts is regulated by GDV1 but may not be linked to sexual commitment.

The Plasmodium falciparum merozoite surface protein MSPDBL2 is a polymorphic antigen targeted by acquired immune responses, and normally expressed in only a minority of mature schizonts. The potential relationship of MSPDBL2 to sexual commitment is examined, as variable mspdbl2 transcript levels and proportions of MSPDBL2-positive mature schizonts in clinical isolates have previously correlated with levels of many sexual stage parasite gene transcripts, although not with the master regulator ap2-g. It is demonstrated that conditional overexpression of the gametocyte development protein GDV1, which promotes sexual commitment, also substantially increases the proportion of MSPDBL2-positive schizonts in culture. Conversely, truncation of the gdv1 gene is shown to prevent any expression of MSPDBL2. However, across diverse P. falciparum cultured lines, the variable proportions of MSPDBL2 positivity in schizonts do not correlate significantly with variable gametocyte conversion rates, indicating it is not involved in sexual commitment. Confirming this, examining a line with endogenous hemagglutinin-tagged AP2-G showed that the individual schizonts expressing MSPDBL2 are mostly different from those expressing AP2-G. Using a selection-linked integration system, modified P. falciparum lines were engineered to express an intact or disrupted version of MSPDBL2, showing the protein is not required for sexual commitment or early gametocyte development. Asexual parasite multiplication rates were also not affected by expression of either intact or disrupted MSPDBL2 in a majority of schizonts. Occurring alongside sexual commitment, the role of the discrete MSPDBL2-positive schizont subpopulation requires further investigation in natural infections where it is under immune selection. IMPORTANCE: Malaria parasites in the blood are remarkably variable, able to switch antigenic targets so they may survive within humans who have already developed specific immune responses. This is one of the challenges in developing vaccines against malaria. MSPDBL2 is a target of naturally acquired immunity expressed in minority proportions of schizonts, the end stages of each 2-day replication cycle in red blood cells which contain merozoites prepared to invade new red blood cells. Results show that the proportion of schizonts expressing MSPDBL2 is positively controlled by the expression of the regulatory gametocyte development protein GDV1. It was previously known that expression of GDV1 leads to increased expression of AP2-G which causes parasites to switch to sexual development, so a surprising finding here is that MSPDBL2-positive parasites are mostly distinct from those that express AP2-G. This discrete antigenic subpopulation of mostly asexual parasites is regulated alongside sexually committed parasites, potentially enabling survival under stress conditions.