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As progress towards malaria elimination continues, the challenge posed by the parasite species Plasmodium vivax has become more evident. In many regions co-endemic for P. vivax and Plasmodium falciparum, as transmission has declined the proportion of cases due to P. vivax has increased. Novel tools that directly target P. vivax are thus warranted for accelerated elimination. There is currently no advanced vaccine for P. vivax and only a limited number of potential candidates in the pipeline. In this study we aimed to identify promising P. vivax proteins that could be used as part of a subunit vaccination approach. We screened 342 P. vivax protein constructs for their ability to induce IgG antibody responses associated with protection from clinical disease in a cohort of children from Papua New Guinea. This approach has previously been used to successfully identify novel candidates. We were able to confirm previous results from our laboratory identifying the proteins reticulocyte binding protein 2b and StAR-related lipid transfer protein, as well as at least four novel candidates with similar levels of predicted protective efficacy. Assessment of these P. vivax proteins in further studies to confirm their potential and identify functional mechanisms of protection against clinical disease are warranted.

Pregnant women have increased susceptibility to Plasmodium falciparum malaria and acquire protective antibodies over successive pregnancies. Most studies that investigated malaria antibody responses in pregnant women are from high transmission areas in sub-Saharan Africa, while reports from Latin America are scarce and inconsistent. The present study sought to explore the development of antibodies against P. falciparum and Plasmodium vivax antigens in pregnant women living in a low transmission area in the Brazilian Amazon.In a prospective cohort study, plasma samples from 408 pregnant women (of whom 111 were infected with P. falciparum, 96 had infections with P. falciparum and P. vivax, and 201 had no Plasmodium infection) were used to measure antibody levels. Levels of IgG and opsonizing antibody to pregnancy-specific variant surface antigens (VSAs) on infected erythrocytes (IEs), 10 recombinant VAR2CSA Duffy binding like (DBL domains), 10 non-pregnancy-specific P. falciparum merozoite antigens, and 10 P. vivax antigens were measured by flow cytometry, ELISA, and multiplex assays. Antibody levels and seropositivity among the groups were compared.Antibodies to VSAs on P. falciparum IEs were generally low but were higher in currently infected women and women with multiple P. falciparum episodes over pregnancy. Many women (21%-69%) had antibodies against each individual VAR2CSA DBL domain, and antibodies to DBLs correlated with each other (r ≥ 0.55, p 0.0001), but not with antibody to VSA or history of infection. Infection with either malaria species was associated with higher seropositivity rate for antibodies against P. vivax proteins, adjusted odds ratios (95% CI) ranged from 5.6 (3.2, 9.7), p 0.0001 for PVDBPII-Sal1 to 15.7 (8.3, 29.7), p 0.0001 for PvTRAg_2.Pregnant Brazilian women had low levels of antibodies to pregnancy-specific VSAs that increased with exposure. They frequently recognized both VAR2CSA DBL domains and P. vivax antigens, but only the latter varied with infection. Apparent antibody prevalence is highly dependent on the assay platform used.

Serological markers are a promising tool for surveillance and targeted interventions for Plasmodium vivax malaria. P. vivax is closely related to the zoonotic parasite P. knowlesi, which also infects humans. P. vivax and P. knowlesi are co-endemic across much of South East Asia, making it important to design serological markers that minimize cross-reactivity in this region. To determine the degree of IgG cross-reactivity against a panel of P. vivax serological markers, we assayed samples from human patients with P. knowlesi malaria. IgG antibody reactivity is high against P. vivax proteins with high sequence identity with their P. knowlesi ortholog. IgG reactivity peaks at 7 days post-P. knowlesi infection and is short-lived, with minimal responses 1 year post-infection. We designed a panel of eight P. vivax proteins with low levels of cross-reactivity with P. knowlesi. This panel can accurately classify recent P. vivax infections while reducing misclassification of recent P. knowlesi infections.

A more sensitive surveillance tool is needed to identify

Purpose Pharmacogenes have an influence on biotransformation pathway and clinical outcome of primaquine and chloroquine which are often prescribed to treat Plasmodium vivax infection. Genetic variation may impact enzyme activity and/or transporter function and thereby contribute to relapse. The aim of the study was to assess allele, genotype frequencies and the association between pharmacogenes variation and primaquine response in Thai patients infected with Plasmodium vivax. Patients and methods Fifty-one patients were genotyped for 74 variants in 18 genes by Sequenom MassARRAY® and Taqman® SNP Real-Time PCR. Results SNP frequencies were not significantly different between relapse (n=4) and non-relapse (n=47) patients. However, the CYP2C19 c.681G>A, the frequency of the A-allele that defines the non-functional CYP2C19*2 haplotype was significantly higher compared to the G-allele (OR=5.14, p=0.021). Patients heterozygous for ABCG2 c.421C>A had a higher odds ratio (OR=8.75, p=0.071) and the frequency of the G-allele of UGT2B7 c.372G>A was higher compared to the A-allele (OR=3.75, p=0.081). CYP2C19, ABCG2 and UGT2B7 emerged as potential high priority genes. Conclusion Decreased activity of CYP2C19, ABCG2 and UGT2B7 in combination with CYP2D6 intermediate or poor metabolizer status may expose patients to a higher risk of Plasmodium vivax relapse. Further investigations are warranted to substantiate these findings.

Background Pregnant women have increased susceptibility to Plasmodium falciparum malaria and acquire protective antibodies over successive pregnancies. Most studies that investigated malaria antibody responses in pregnant women are from high transmission areas in sub-Saharan Africa, while reports from Latin America are scarce and inconsistent. The present study sought to explore the development of antibodies against P. falciparum and Plasmodium vivax antigens in pregnant women living in a low transmission area in the Brazilian Amazon. Methods In a prospective cohort study, plasma samples from 408 pregnant women (of whom 111 were infected with P. falciparum, 96 had infections with P. falciparum and P. vivax, and 201 had no Plasmodium infection) were used to measure antibody levels. Levels of IgG and opsonizing antibody to pregnancy-specific variant surface antigens (VSAs) on infected erythrocytes (IEs), 10 recombinant VAR2CSA Duffy binding like (DBL domains), 10 non-pregnancy-specific P. falciparum merozoite antigens, and 10 P. vivax antigens were measured by flow cytometry, ELISA, and multiplex assays. Antibody levels and seropositivity among the groups were compared. Results Antibodies to VSAs on P. falciparum IEs were generally low but were higher in currently infected women and women with multiple P. falciparum episodes over pregnancy. Many women (21%-69%) had antibodies against each individual VAR2CSA DBL domain, and antibodies to DBLs correlated with each other (r ≥ 0.55, p P. vivax proteins, adjusted odds ratios (95% CI) ranged from 5.6 (3.2, 9.7), p Conclusions Pregnant Brazilian women had low levels of antibodies to pregnancy-specific VSAs that increased with exposure. They frequently recognized both VAR2CSA DBL domains and P. vivax antigens, but only the latter varied with infection. Apparent antibody prevalence is highly dependent on the assay platform used.

Multiplexed bead-based assays that use Luminex® xMAP® technology have become popular for measuring antibodies against proteins of interest in many fields, including malaria and more recently SARS-CoV-2/COVID-19. There are currently two formats that are widely used: non-magnetic beads or magnetic beads. Data are lacking regarding the comparability of results obtained using these two types of beads, and for assays run on different instruments. Whilst non-magnetic beads can only be run on flow-based instruments (such as the Luminex® 100/200™ or Bio-Plex® 200), magnetic beads can be run on both these and the newer MAGPIX® instruments. In this study we utilized a panel of purified recombinant Plasmodium vivax proteins and samples from malaria-endemic areas to measure P. vivax-specific IgG responses using different combinations of beads and instruments. We directly compared: i) non-magnetic versus magnetic beads run on a Bio-Plex® 200, ii) magnetic beads run on the Bio-Plex® 200 versus MAGPIX® and iii) non-magnetic beads run on a Bio-Plex® 200 versus magnetic beads run on the MAGPIX®. We also performed an external comparison of our optimized assay. We observed that IgG antibody responses, measured against our panel of P. vivax proteins, were moderately-strongly correlated in all three of our comparisons (pearson r>0.5 for 18/19 proteins), however higher amounts of protein were required for coupling to magnetic beads. Our external comparison indicated that results generated in different laboratories using the same coupled beads are also highly comparable (pearson r>0.7), particularly if a reference standard curve is used.

A more sensitive surveillance tool is needed to identify Plasmodium vivax infections for treatment and to accelerate malaria elimination efforts. To address this challenge, our laboratory has developed an eight-antigen panel that detects total IgG as serological markers of P. vivax exposure within the prior 9 months. The value of these markers has been established for use in areas with low transmission. In moderate–high transmission areas, there is evidence that total IgG is more long-lived than in areas with low transmission, resulting in poorer performance of these markers in these settings. Antibodies that are shorter-lived may be better markers of recent infection for use in moderate–high transmission areas. Using a multiplex assay, the antibody temporal kinetics of total IgG, IgG1, IgG3, and IgM against 29 P. vivax antigens were measured over 36 weeks following asymptomatic P. vivax infection in Papua New Guinean children (n = 31), from an area with moderate–high transmission intensity. IgG3 declined faster to background than total IgG, IgG1, and IgM. Based on these kinetics, IgG3 performance was then assessed for classifying recent exposure in a cohort of Peruvian individuals (n = 590; age 3–85 years) from an area of moderate transmission intensity. Using antibody responses against individual antigens, the highest performance of IgG3 in classifying recent P. vivax infections in the prior 9 months was to one of the Pv-fam-a proteins assessed (PVX_125728) (AUC = 0.764). Surprisingly, total IgG was overall a better marker of recent P. vivax infection, with the highest individual classification performance to RBP2b1986-2653 (PVX_094255) (AUC = 0.838). To understand the acquisition of IgG3 in this Peruvian cohort, relevant epidemiological factors were explored using a regression model. IgG3 levels were positively associated with increasing age, living in an area with (relatively) higher transmission intensity, and having three or more PCR-detected blood-stage P. vivax infections within the prior 13 months. Overall, we found that IgG3 did not have high accuracy for detecting recent exposure to P. vivax in the Peruvian cohort, with our data suggesting that this is due to the high levels of prior exposure required to acquire high IgG3 antibody levels.

SummarySerological exposure markers are a promising tool for surveillance and targeted interventions for Plasmodium vivax malaria. P. vivax is closely related to the zoonotic parasite P. knowlesi, which also infects humans. P. vivax and P. knowlesi are co-endemic across much of South East Asia, making it important to design P. vivax serological markers that minimise cross-reactivity in this region. Our objective was to determine the degree of IgG antibody cross-reactivity against a panel of P. vivax serological markers in samples from human participants with P. knowlesi malaria. We observed higher levels of IgG antibody reactivity against P. vivax proteins that had high levels of sequence identity with their P. knowlesi ortholog. IgG reactivity peaked at 7 days post P. knowlesi infection and was short-lived, with minimal responses detected at 1-year post-infection. Using these data, we designed a panel of 8 P. vivax proteins with low-levels of cross-reactivity with P. knowlesi. This panel was able to accurately classify recent P. vivax infections whilst reducing misclassification of recent P. knowlesi infections.

Background Plasmodium vivax (P. 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. Methods 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® assay and identified protein-specific variation in antibody longevity. Mathematical modelling was used to generate the estimated half-life of antibodies, long-, and short-lived antibody-secreting cells. Results 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. Conclusions 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.

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® 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.

The rotating-crystal magneto-optical detection (RMOD) method has been developed for the rapid and quantitative diagnosis of malaria and tested systematically on various malaria infection models. Very recently, an extended field trial in a high-transmission region of Papua New Guinea demonstrated its great potential for detecting malaria infections, in particular Plasmodium vivax. In the present small-scale field test, carried out in a low-transmission area of Thailand, RMOD confirmed malaria in all samples found to be infected with Plasmodium vivax by microscopy, our reference method. Moreover, the magneto-optical signal for this sample set was typically 1–3 orders of magnitude higher than the cut-off value of RMOD determined on uninfected samples. Based on the serial dilution of the original patient samples, we expect that the method can detect Plasmodium vivax malaria in blood samples with parasite densities as low as $$\sim$$ ∼ 5–10 parasites per microliter, a limit around the pyrogenic threshold of the infection. In addition, by investigating the correlation between the magnitude of the magneto-optical signal, the parasite density and the erythrocytic stage distribution, we estimate the relative hemozoin production rates of the ring and the trophozoite stages of in vivo Plasmodium vivax infections.

Thailand is aiming for malaria elimination by the year 2030. However, the high proportion of asymptomatic infections and the presence of the hidden hypnozoite stage of Plasmodium vivax are impeding these efforts. We hypothesized that a validated surveillance tool utilizing serological markers of recent exposure to P. vivax infection could help to identify areas of ongoing transmission. The objective of this exploratory study was to assess the ability of P. vivax serological exposure markers to detect residual transmission “hot-spots” in Western Thailand. Total IgG levels were measured against a panel of 23 candidate P. vivax serological exposure markers using a multiplexed bead-based assay. A total of 4,255 plasma samples from a cross-sectional survey conducted in 2012 of endemic areas in the Kanchanaburi and Ratchaburi provinces were assayed. We compared IgG levels with multiple epidemiological factors that are associated with an increased risk of P. vivax infection in Thailand, including age, gender, and spatial location, as well as Plasmodium infection status itself. IgG levels to all proteins were significantly higher in the presence of a P. vivax infection (n = 144) (T-test, p < 0.0001). Overall seropositivity rates varied from 2.5% (PVX_097625, merozoite surface protein 8) to 16.8% (PVX_082670, merozoite surface protein 7), with 43% of individuals seropositive to at least 1 protein. Higher IgG levels were associated with older age (>18 years, p < 0.05) and males (17/23 proteins, p < 0.05), supporting the paradigm that men have a higher risk of infection than females in this setting. We used a Random Forests algorithm to predict which individuals had exposure to P. vivax parasites in the last 9-months, based on their IgG antibody levels to a panel of eight previously validated P. vivax proteins. Spatial clustering was observed at the village and regional level, with a moderate correlation between PCR prevalence and sero-prevalence as predicted by the algorithm. Our data provides proof-of-concept for application of such surrogate markers as evidence of recent exposure in low transmission areas. These data can be used to better identify geographical areas with asymptomatic infection burdens that can be targeted in elimination campaigns.

To achieve malaria elimination, new tools are required to explicitly target Plasmodium vivax. Recently, a novel panel of P. vivax proteins were identified and validated as serological markers for detecting recent exposure to P. vivax within the last 9 months. In order to improve the sensitivity and specificity of these markers, immunoglobulin M (IgM) in addition to immunoglobulin G (IgG) antibody responses were compared with a down-selected panel of 20 P. vivax proteins. IgM was tested using archival plasma samples from observational cohort studies conducted in malaria-endemic regions of Thailand and Brazil. IgM responses to these proteins generally had poorer classification performance than IgG.

The rotating-crystal magneto-optical detection (RMOD) method has been developed for the rapid and quantitative diagnosis of malaria and tested systematically on various malaria infection models. Very recently, an extended field trial in a high-transmission region of Papua New Guinea demonstrated its great potential for detecting malaria infections, in particular Plasmodium vivax. In the present small-scale field test, carried out in a low-transmission area of Thailand, RMOD confirmed malaria in all samples found to be infected with Plasmodium vivax by microscopy, our reference method. Moreover, the magneto-optical signal for this sample set was typically 1–3 orders of magnitude higher than the cut-off value of RMOD determined on uninfected samples. Based on the serial dilution of the original patient samples, we expect that the method can detect Plasmodium vivax malaria in blood samples with parasite densities as low as ∼ 5 − 10 parasites per microliter, a limit around the pyrogenic threshold of the infection. In addition, by investigating the correlation between the magnitude of the magneto-optical signal, the parasite density and the erythrocytic stage distribution, we estimate the relative hemozoin production rates of the ring and the trophozoite stages of in vivo Plasmodium vivax infections.

Thailand is aiming for malaria elimination by the year 2030. However, the high proportion of asymptomatic infections and the presence of the hidden hypnozoite stage of Plasmodium vivax are impeding these efforts. We hypothesized that a validated surveillance tool utilizing serological markers of recent exposure to P. vivax infection could help to identify areas of ongoing transmission. The objective of this exploratory study was to assess the ability of P. vivax serological exposure markers to detect residual transmission ‘hot-spots’ in Western Thailand. Total IgG levels were measured against a panel of 23 candidate P. vivax serological exposure markers using a multiplexed bead-based assay. A total of 4255 plasma samples from a cross-sectional survey conducted in 2012 of endemic areas in the Kanchanaburi and Ratchaburi provinces were assayed. We compared IgG levels with multiple epidemiological factors that are associated with an increased risk of P. vivax infection in Thailand, including age, gender and spatial location, as well as Plasmodium infection status itself. IgG levels to all proteins were significantly higher in the presence of a P. vivax infection (n=144) (t test, p18 years, pP. vivax parasites in the last 9-months, based on their IgG antibody levels to a panel of 8 previously validated P. vivax proteins. Spatial clustering was observed at the village and regional level, with a moderate correlation between PCR prevalence and sero-prevalence as predicted by the algorithm. Our data provides proof-of-concept for application of such surrogate markers as evidence of recent exposure in low transmission areas. These data can be used to better identify geographical areas with asymptomatic infection burdens that can be targeted in elimination campaigns.

Thailand is aiming for malaria elimination by the year 2030. However, the high proportion of asymptomatic infections and the presence of the hidden hypnozoite stage of Plasmodium vivax are impeding these efforts. We hypothesized that a validated surveillance tool utilizing serological markers of recent exposure to P. vivax infection could help to identify areas of ongoing transmission. The objective of this exploratory study was to assess the ability of P. vivax serological exposure markers to detect residual transmission “hot-spots” in Western Thailand. Total IgG levels were measured against a panel of 23 candidate P. vivax serological exposure markers using a multiplexed bead-based assay. A total of 4,255 plasma samples from a cross-sectional survey conducted in 2012 of endemic areas in the Kanchanaburi and Ratchaburi provinces were assayed. We compared IgG levels with multiple epidemiological factors that are associated with an increased risk of P. vivax infection in Thailand, including age, gender, and spatial location, as well as Plasmodium infection status itself. IgG levels to all proteins were significantly higher in the presence of a P. vivax infection (n = 144) (T-test, p < 0.0001). Overall seropositivity rates varied from 2.5% (PVX_097625, merozoite surface protein 8) to 16.8% (PVX_082670, merozoite surface protein 7), with 43% of individuals seropositive to at least 1 protein. Higher IgG levels were associated with older age (>18 years, p < 0.05) and males (17/23 proteins, p < 0.05), supporting the paradigm that men have a higher risk of infection than females in this setting. We used a Random Forests algorithm to predict which individuals had exposure to P. vivax parasites in the last 9-months, based on their IgG antibody levels to a panel of eight previously validated P. vivax proteins. Spatial clustering was observed at the village and regional level, with a moderate correlation between PCR prevalence and sero-prevalence as predicted by the algorithm. Our data provides proof-of-concept for application of such surrogate markers as evidence of recent exposure in low transmission areas. These data can be used to better identify geographical areas with asymptomatic infection burdens that can be targeted in elimination campaigns.

Plasmodium vivax is the most widespread causative agent of human malaria in the world. Despite the ongoing implementation of malaria control programs, the rate of case reduction has declined over the last 5 years. Hence, surveillance of malaria transmission should be in place to identify and monitor areas that require intensified malaria control interventions. Serological tools may offer additional insights into transmission intensity over parasite and entomological measures, especially as transmission levels decline. Antibodies can be detected in the host system for months to even years after parasite infections have been cleared from the blood, enabling malaria exposure history to be captured. Because the Plasmodium parasite expresses more than 5000 proteins, it is important to a) understand antibody longevity following infection and b) measure antibodies to more than one antigen in order to accurately inform on the exposure and/or immune status of populations. This review summarises current practices for surveillance of P. vivax malaria, the current state of research into serological exposure markers and their potential role for accelerating malaria elimination, and discusses further studies that need to be undertaken to see such technology implemented in malaria-endemic areas.

Multiplexed bead-based assays that use Luminex xMAP® technology have become popular for measuring antibodies against proteins of interest in many fields, including malaria and more recently SARS-CoV-2/COVID-19. There are currently two formats that are widely used: non-magnetic beads or magnetic beads. Data is lacking regarding the comparability of results obtained using these two types of beads, and for assays run on different instruments. Whilst non-magnetic beads can only be run on flow-based instruments (such as the Luminex® 100/200™ or Bio-Plex® 200), magnetic beads can be run on both these and the newer MAGPIX® instruments. In this study we utilized a panel of purified recombinant Plasmodium vivax proteins and samples from malaria-endemic areas to measure P. vivax-specific IgG responses using different combinations of beads and instruments. We directly compared: i) non-magnetic versus magnetic beads run on a Bio-Plex® 200, ii) magnetic beads run on the Bio-Plex® 200 versus MAGPIX® and iii) non-magnetic beads run on a Bio-Plex® 200 versus magnetic beads run on the MAGPIX®. We also performed an external validation of our optimized assay. We observed that IgG antibody responses, measured against our panel of P. vivax proteins, were strongly correlated in all three of our comparisons, however higher amounts of protein were required for coupling to magnetic beads. Our external validation indicated that results generated in different laboratories using the same coupled beads are also highly comparable, particularly if a reference standard curve is used.

To achieve malaria elimination, new tools are required to explicitly target Plasmodium vivax. Recently, a novel panel of P. vivax proteins were identified and validated as serological markers for detecting recent exposure to P. vivax within the last 9 months. In order to improve the sensitivity and specificity of these markers, IgM in addition to IgG antibody responses were assessed to a down-selected panel of 20 P. vivax proteins. IgM was tested using archival plasma samples from observational cohort studies conducted in malaria-endemic regions of Thailand and Brazil. IgM responses to these proteins generally had poorer classification performance than IgG.

BackgroundAntibody responses as serological markers ofPlasmodium vivaxinfection have been shown to correlate with exposure, but little is known about the other factors that affect antibody responses in naturally infected people from endemic settings. To address this question, we studied IgG responses to novel serological exposure markers (SEMs) ofP. vivaxin three settings with different transmission intensity.MethodologyWe validated a panel of 34 SEMs in a Peruvian cohort with up to three years’ longitudinal follow-up using a multiplex platform and compared results to data from cohorts in Thailand and Brazil. Linear regression models were used to characterize the association between antibody responses and age, the number of detected blood-stage infections during follow-up, and time since previous infection. Receiver Operating Characteristic (ROC) analysis was used to test the performance of SEMs to identifyP. vivaxinfections in the previous 9 months.Principal findingsAntibody titers were associated with age, the number of blood-stage infections, and time since previousP. vivaxinfection in all three study sites. The association between antibody titers and time since previousP. vivaxinfection was stronger in the low transmission settings of Thailand and Brazil compared to the higher transmission setting in Peru. Of the SEMs tested, antibody responses to RBP2b had the highest performance for classifying recent exposure in all sites, with area under the ROC curve (AUC) = 0.83 in Thailand, AUC = 0.79 in Brazil, and AUC = 0.68 in Peru.ConclusionsIn low transmission settings,P. vivaxSEMs can accurately identify individuals with recent blood-stage infections. In higher transmission settings, the accuracy of this approach diminishes substantially. We recommend usingP. vivaxSEMs in low transmission settings pursuing malaria elimination, but they are likely to be less effective in high transmission settings focused on malaria control.Author SummaryPlasmodium vivaxstill poses a threat in many countries due to its ability to cause recurrent infections. Key to achieving the goal of malaria elimination is the ability to quickly detect and treat carriers of relapsing parasites. Failing to identify this transmission reservoir will hinder progress towards malaria elimination. Recently, novel serological markers of recent exposure toP. vivax(SEM) have been developed and validated in low transmission settings. It is still poorly understood what factors affect the antibody response to these markers when evaluated in contrasting endemic contexts. To determine the factors that influence the antibody response to SEM, we compared the antibody levels in three sites with different transmission intensity: Thailand (low), Brazil (moderate) and Peru (high). In this study, we found that transmission intensity plays a key role in the acquisition of the antibody repertoire toP.vivax. In highly endemic sites, it is likely that immunological memory resulting from a constant and sustained exposure will impact the performance of SEMs to detect individuals with recent exposure toP.vivax. In summary, SEMs that perform well in low transmission sites do not perform as well in high transmission regions.

BackgroundInfection with SARS-CoV-2 induces an antibody response targeting multiple antigens that changes over time. This complexity presents challenges and opportunities for serological diagnostics.MethodsA multiplex serological assay was developed to measure IgG and IgM antibody responses to seven SARS-CoV-2 spike or nucleoprotein antigens, two antigens for the nucleoproteins of the 229E and NL63 seasonal coronaviruses, and three non-coronavirus antigens. Antibodies were measured in serum samples from patients in French hospitals with RT-qPCR confirmed SARS-CoV-2 infection (n= 259), and negative control serum samples collected before the start of the SARS-CoV-2 epidemic (n= 335). A random forests algorithm was trained with the multiplex data to classify individuals with previous SARS-CoV-2 infection. A mathematical model of antibody kinetics informed by prior information from other coronaviruses was used to estimate time-varying antibody responses and assess the potential sensitivity and classification performance of serological diagnostics during the first year following symptom onset. A statistical estimator is presented that can provide estimates of seroprevalence in very low transmission settings.ResultsIgG antibody responses to trimeric Spike protein identified individuals with previous RT-qPCR confirmed SARS-CoV-2 infection with 91.6% sensitivity (95% confidence interval (CI); 87.5%, 94.5%) and 99.1% specificity (95% CI; 97.4%, 99.7%). Using a serological signature of IgG and IgM to multiple antigens, it was possible to identify infected individuals with 98.8% sensitivity (95% CI; 96.5%, 99.6%) and 99.3% specificity (95% CI; 97.6%, 99.8%). Informed by prior data from other coronaviruses, we estimate that one year following infection a monoplex assay with optimal anti-StriIgG cutoff has 88.7% sensitivity (95% CI: 63.4%, 97.4%), and that a multiplex assay can increase sensitivity to 96.4% (95% CI: 80.9%, 100.0%). When applied to population-level serological surveys, statistical analysis of multiplex data allows estimation of seroprevalence levels less than 1%, below the false positivity rate of many other assays.ConclusionSerological signatures based on antibody responses to multiple antigens can provide accurate and robust serological classification of individuals with previous SARS-CoV-2 infection. This provides potential solutions to two pressing challenges for SARS-CoV-2 serological surveillance: classifying individuals who were infected greater than six months ago, and measuring seroprevalence in serological surveys in very low transmission settings.

International audience; A major gap in the Plasmodium vivax elimination toolkit is the identification of individuals carrying clinically silent and undetectable liver-stage parasites, called hypnozoites. This study developed a panel of serological exposure markers capable of classifying individuals with recent P. vivax infections who have a high likelihood of harboring hypnozoites. We measured IgG antibody responses to 342 P. vivax proteins in longitudinal clinical cohorts conducted in Thailand and Brazil and identified candidate serological markers of exposure. Candidate markers were validated using samples from year-long observational cohorts conducted in Thailand, Brazil and the Solomon Islands and antibody responses to eight P. vivax proteins classified P. vivax infections in the previous 9 months with 80% sensitivity and specificity. Mathematical models demonstrate that a serological testing and treatment strategy could reduce P. vivax prevalence by 59-69%. These eight antibody responses can serve as a biomarker, identifying individuals who should be targeted with anti-hypnozoite therapy.

Serology tests are extremely useful for assessing whether a person has been infected with a pathogen. Since the onset of the COVID-19 pandemic, measurement of anti-SARS-CoV-2-specific antibodies has been considered an essential tool in identifying seropositive individuals and thereby understanding the extent of transmission in communities. The Luminex system is a bead-based technology that has the capacity to assess multiple antigens simultaneously using very low sample volumes and is ideal for high-throughput studies. We have adapted this technology to develop a COVID-19 multi-antigen serological assay. This protocol described here carefully outlines recommended steps to optimize and establish this method for COVID-19-specific antibody measurement in plasma and in saliva. However, the protocol can easily be customized and thus the assay is broadly applicable to measure antibodies to other pathogens.

Despite promising progress in malaria vaccine development in recent years, an efficacious subunit vaccine against Plasmodium falciparum remains to be licensed and deployed. Cell-mediated protection from liver-stage malaria relies on a sufficient number of antigen-specific T cells reaching the liver during the time that parasites are present. A single vaccine expressing two antigens could potentially increase both the size and breadth of the antigen-specific response while halving vaccine production costs., Despite promising progress in malaria vaccine development in recent years, an efficacious subunit vaccine against Plasmodium falciparum remains to be licensed and deployed. Cell-mediated protection from liver-stage malaria relies on a sufficient number of antigen-specific T cells reaching the liver during the time that parasites are present. A single vaccine expressing two antigens could potentially increase both the size and breadth of the antigen-specific response while halving vaccine production costs. In this study, we investigated combining two liver-stage antigens, P. falciparum LSA1 (PfLSA1) and PfLSAP2, and investigated the induction of protective efficacy by coadministration of single-antigen vectors or vaccination with dual-antigen vectors, using simian adenovirus and modified vaccinia virus Ankara vectors. The efficacy of these vaccines was assessed in mouse malaria challenge models using chimeric P. berghei parasites expressing the relevant P. falciparum antigens and challenging mice at the peak of the T cell response. Vaccination with a combination of the single-antigen vectors expressing PfLSA1 or PfLSAP2 was shown to improve protective efficacy compared to vaccination with each single-antigen vector alone. Vaccination with dual-antigen vectors expressing both PfLSA1 and PfLSAP2 resulted in responses to both antigens, particularly in outbred mice, and most importantly, the efficacy was equivalent to that of vaccination with a mixture of single-antigen vectors. Based on these promising data, dual-antigen vectors expressing PfLSA1 and PfLSAP2 will now proceed to manufacturing and clinical assessment under good manufacturing practice (GMP) guidelines.

SUMMARYPlasmodium vivaxis the most geographically widespread of the malaria parasites causing human disease, yet it is comparatively understudied compared withPlasmodium falciparum.In this article we review what is known about naturally acquired immunity toP. vivax, and importantly, how this differs to that acquired againstP. falciparum.Immunity to clinicalP. vivaxinfection is acquired more quickly than toP. falciparum, and evidence suggests humans in endemic areas also have a greater capacity to mount a successful immunological memory response to this pathogen. Both of these factors give promise to the idea of a successfulP. vivaxvaccine. We review what is known about both the cellular and humoral immune response, including the role of cytokines, antibodies, immunoregulation, immune memory and immune dysfunction. Furthermore, we discuss where the future lies in terms of advancing our understanding of naturally acquired immunity toP. vivax, through the use of well-designed longitudinal epidemiological studies and modern tools available to immunologists.

In order to accelerate towards malaria elimination, improved targeting of limited resources is essential. A major gap in our elimination toolkit forPlasmodium vivaxmalaria is the identification of individuals carrying arrested liver stages, called hypnozoites. These clinically silent but frequently relapsing hypnozoites are key toP. vivaxpersistence. Whilst hypnozoites cannot be directly detected, individuals who have had recent exposure toP. vivaxand have not been treated are likely to harbor these parasites. By measuring IgG antibody responses to over 300P. vivaxproteins, a panel of serological markers capable of detecting exposure toP. vivaxinfections in the prior 9-month period was identified and validated. Using antibody responses to 8P. vivaxproteins, 80% sensitivity and specificity for detecting recent infections were achieved in three independent studies conducted in Thailand, Brazil and the Solomon Islands. As these individuals have a high likelihood of harboring hypnozoites, the suite of these 8 antibody responses can serve as biomarkers for the identification of individuals who should be targeted for treatment with liver-stage drugs such as primaquine and tafenoquine in mass drug administration programs aimed at controlling and eliminatingP. vivaxmalaria.One Sentence SummaryThe manuscript describes identification and validation of a novel panel of P. vivax proteins that can be used to detect recent exposure to P. vivax infections within the prior 9 months.

BACKGROUND: In malaria endemic populations, complex patterns of Plasmodium vivax and Plasmodium falciparum blood-stage infection dynamics may be observed. Genotyping samples from longitudinal cohort studies for merozoite surface protein (msp) variants increases the information available in the data, allowing multiple infecting parasite clones in a single individual to be identified. msp genotyped samples from two longitudinal cohorts in Papua New Guinea (PNG) and Thailand were analysed using a statistical model where the times of acquisition and clearance of each clone in every individual were estimated using a process of data augmentation. RESULTS: For the populations analysed, the duration of blood-stage P. falciparum infection was estimated as 36 (95% Credible Interval (CrI): 29, 44) days in PNG, and 135 (95% CrI 94, 191) days in Thailand. Experiments on simulated data indicated that it was not possible to accurately estimate the duration of blood-stage P. vivax infections due to the lack of identifiability between a single blood-stage infection and multiple, sequential blood-stage infections caused by relapses. Despite this limitation, the method and data point towards short duration of blood-stage P. vivax infection with a lower bound of 24 days in PNG, and 29 days in Thailand. On an individual level, P. vivax recurrences cannot be definitively classified into re-infections, recrudescences or relapses, but a probabilistic relapse phenotype can be assigned to each P. vivax sample, allowing investigation of the association between epidemiological covariates and the incidence of relapses. CONCLUSION: The statistical model developed here provides a useful new tool for in-depth analysis of malaria data from longitudinal cohort studies, and future application to data sets with multi-locus genotyping will allow more detailed investigation of infection dynamics.

A large research effort is currently underway to find an effective and affordable malaria vaccine. Tools that enable the rapid evaluation of protective immune responses are essential to vaccine development as they can provide selection criteria to rank order vaccine candidates. In this study we have revisited the Inhibition of Sporozoite Invasion (ISI) assay to assess the ability of antibodies to inhibit sporozoite infection of hepatocytes. By using GFP expressing sporozoites of the rodent parasite P. berghei we are able to robustly quantify parasite infection of hepatocyte cell lines by flow cytometry. In conjunction with recently produced transgenic P. berghei parasites that express P. falciparum sporozoite antigens, we have been able to use this assay to measure antibody mediated inhibition of sporozoite invasion against one of the lead malaria antigens P. falciparum CSP. By combining chimeric rodent parasites expressing P. falciparum antigens and a flow cytometric readout of infection, we are able to robustly assess vaccine-induced antibodies, from mice, rhesus macaques and human clinical trials, for their functional ability to block sporozoite invasion of hepatocytes.

BACKGROUND: Thailand is aiming to eliminate malaria by the year 2024. Plasmodium vivax has now become the dominant species causing malaria within the country, and a high proportion of infections are asymptomatic. A better understanding of antibody dynamics to P. vivax antigens in a low-transmission setting, where acquired immune responses are poorly characterized, will be pivotal for developing new strategies for elimination, such as improved surveillance methods and vaccines. The objective of this study was to characterize total IgG antibody levels to 11 key P. vivax proteins in a village of western Thailand. METHODS: Plasma samples from 546 volunteers enrolled in a cross-sectional survey conducted in 2012 in Kanchanaburi Province were utilized. Total IgG levels to 11 different proteins known or predicted to be involved in reticulocyte binding or invasion (ARP, GAMA, P41, P12, PVX_081550, and five members of the PvRBP family), as well as the leading pre-erythrocytic vaccine candidate (CSP) were measured using a multiplexed bead-based assay. Associations between IgG levels and infection status, age, and spatial location were explored. RESULTS: Individuals from a low-transmission region of western Thailand reacted to all 11 P. vivax recombinant proteins. Significantly greater IgG levels were observed in the presence of a current P. vivax infection, despite all infected individuals being asymptomatic. IgG levels were also higher in adults (18 years and older) than in children. For most of the proteins, higher IgG levels were observed in individuals living closer to the Myanmar border and further away from local health services. CONCLUSIONS: Robust IgG responses were observed to most proteins and IgG levels correlated with surrogates of exposure, suggesting these antigens may serve as potential biomarkers of exposure, immunity, or both.

Plasmodium vivax remains an important cause of malaria in South America and the Asia-Pacific. Naturally acquired antibody responses against multiple P. vivax proteins have been described in numerous countries, however, direct comparison of these responses has been difficult with different methodologies employed. We measured antibody responses against 307 P. vivax proteins at the time of P. vivax infection, and at 2–3 later time-points in three countries. We observed that seropositivity rates at the time of infection were highest in Thailand, followed by Brazil then PNG, reflecting the level of antigenic input. The majority of sero-reactive antigens in all sites induced short-lived antibody responses with estimated half-lives of less than 6 months, although there was a trend towards longer-lived responses in PNG children. Despite these differences, IgG seropositivity rates, magnitude and longevity were highly and significantly rank-correlated between the different regions, suggesting such features are reflective of the individual protein., Author summary In the pursuit of eliminating all species of malaria, Plasmodium vivax presents one of the most substantial challenges, particularly in countries in Asia, the Western-Pacific and South America. This is primarily due to the ability of P. vivax to cause relapse infections months to years after the initial infectious bite. In areas with low levels of malaria transmission, serology has become an increasingly useful tool for surveillance, as anti-Plasmodium antibodies can be detected in individuals long after blood-stage parasites have cleared. In this study, we provide a detailed characterisation of the antibody response generated following P. vivax infection by measuring antibodies to over 300 P. vivax antigens in three different populations in Thailand, Brazil and Papua New Guinea. The individuals in these populations were followed for up to nine months allowing us to estimate the rate at which antibodies decay over time. This improved understanding of the magnitude and dynamics of the antibody response, validated in multiple populations, will contribute to the development of serological surveillance tools needed for enhanced control and elimination of P. vivax.

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Plasmodium vivax is now the dominant Plasmodium species causing malaria in Thailand, yet little is known about naturally acquired immune responses to this parasite in this low-transmission region. The preerythrocytic stage of the P. vivax life cycle is considered an excellent target for a malaria vaccine, and in this study, we assessed the stability of the seropositivity and the magnitude of IgG responses to three different preerythrocytic P. vivax proteins in two groups of adults from a region of western Thailand where malaria is endemic. These individuals were enrolled in a yearlong cohort study, which comprised one group that remained P. vivax free (by quantitative PCR [qPCR] detection, n = 31) and another that experienced two or more blood-stage P. vivax infections during the year of follow up ( n = 31). Despite overall low levels of seropositivity, IgG positivity and magnitude were long-lived over the 1-year period in the absence of qPCR-detectable blood-stage P. vivax infections. In contrast, in the adults with two or more P. vivax infections during the year, IgG positivity was maintained, but the magnitude of the response to P. vivax circumsporozoite protein 210 (CSP210) decreased over time. These findings demonstrate that long-term humoral immunity can develop in low-transmission regions.

Efforts are under way to improve the efficacy of subunit malaria vaccines through assessments of new adjuvants, vaccination platforms, and antigens. In this study, we further assessed the Plasmodium falciparum antigen upregulated in infective sporozoites 3 (PfUIS3) as a vaccine candidate. PfUIS3 was expressed in the viral vectors chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) and used to immunize mice in a prime-boost regimen. We previously demonstrated that this regimen could provide partial protection against challenge with chimeric P. berghei parasites expressing PfUIS3. We now show that ChAd63-MVA PfUIS3 can also provide partial cross-species protection against challenge with wild-type P. berghei parasites. We also show that PfUIS3-specific cellular memory responses could be recalled in human volunteers exposed to P. falciparum parasites in a controlled human malaria infection study. When ChAd63-MVA PfUIS3 was coadministered with the vaccine candidate P. falciparum thrombospondin-related adhesion protein (PfTRAP) expressed in the ChAd63-MVA system, there was no significant change in immunogenicity to either vaccine. However, when mice were challenged with double chimeric P. berghei-P. falciparum parasites expressing both PfUIS3 and PfTRAP, vaccine efficacy was improved to 100% sterile protection. This synergistic effect was evident only when the two vaccines were mixed and administered at the same site. We have therefore demonstrated that vaccination with PfUIS3 can induce a consistent delay in patent parasitemia across mouse strains and against chimeric parasites expressing PfUIS3 as well as wild-type P. berghei; when this vaccine is combined with another partially protective regimen (ChAd63-MVA PfTRAP), complete protection is induced.

Since the demonstration of sterile protection afforded by injection of irradiated sporozoites, CD8+ T cells have been shown to play a significant role in protection from liver-stage malaria. This is, however, dependent on the presence of an extremely high number of circulating effector cells, thought to be necessary to scan, locate, and kill infected hepatocytes in the short time that parasites are present in the liver. We used an adoptive transfer model to elucidate the kinetics of the effector CD8+ T cell response in the liver following Plasmodium berghei sporozoite challenge. Although effector CD8+ T cells require

Approximately 1.4 billion people are at some risk of malaria infection in the South East Asia region, with 352 million at high risk. The most common and effective malaria vector control strategies currently in use are based on insecticides: indoor residual spraying (IRS) and long-lasting insecticide-treated nets. However, these interventions were designed for indoor-biting vectors, and hence are not necessarily effective in all regions. Although there are many nongenetic tools for vector control, a major limitation is the research-based evaluation of efficiency of those tools in many areas. Another limitation for research of malaria vectors in Asia is due to the high complexity of many vector species and the difficulty to maintain most of the important vectors in the laboratory, as it is very labor intensive. Here, we review the ecological and behavioral characteristics of only the most important anthropophilic vectors within this region: Anopheles culicifacies , Anopoheles fluviatilis , and Anopheles stephensi across the Indian subcontinent; and Anopheles dirus and Anopheles minimus within Southeast Asia. The challenges in applying genetic control methods to malaria vectors in Asia will be similar to the other nongenetic tools that have been used to control vectors in this region: species complexity, multiple vectors, and potential vectors in each endemic area and only a few species that can be successfully maintained in the laboratory.

Substantial protection can be provided against the pre-erythrocytic stages of malaria by vaccination first with an adenoviral and then with an modified vaccinia virus Ankara (MVA) poxviral vector encoding the same ME.TRAP transgene. We investigated whether the two vaccine components adenovirus (Ad) and MVA could be coinjected as a mixture to enhance protection against malaria. A single-shot mixture at specific ratios of Ad and MVA (AdMVA) enhanced CD8 ++ T cell-dependant protection of mice against challenge with Plasmodium berghei. Moreover, the degree of protection could be enhanced after homologous boosting with the same AdMVA mixture to levels comparable with classic heterologous Ad prime-MVA boost regimes. The mixture increased transgene-specific responses while decreasing the CD8++ T cell antivector immunity compared to each vector used alone, particularly against the MVA backbone. Mixed vector immunization led to increased early circulating interferon-γ (IFN-γ) response levels and altered transcriptional microarray profiles. Furthermore, we found that sequential immunizations with the AdMVA mixture led to consistent boosting of the transgene-specific CD8++ response for up to three mixture immunizations, whereas each vector used alone elicited progressively lower responses. Our findings offer the possibility of simplifying the deployment of viral vectors as a single mixture product rather than in heterologous prime-boost regimens. © The American Society of Gene and Cell Therapy.

Malaria is a disease that infects over 500 million people, causing at least 1 million deaths every year, with the majority occurring in developing countries. The current antimalarial arsenal is becoming dulled due to the rapid rate of resistance of the parasite. However, in populations living in malaria-endemic regions there are many examples of genetic-based resistance to the severe effects of the parasite Plasmodium. Defining the genetic factors behind host resistance has been an area of great scientific interest over the last few decades; this review summarizes the current knowledge of the genetic loci involved. Perhaps the lessons learned from the natural variation in both the human populations and experimental mouse models of infection may pave the way for novel resistance-proof antimalarials.

The development of a highly efficacious and durable vaccine for malaria remains a top priority for global health researchers. Despite the huge rise in recognition of malaria as a global health problem and the concurrent rise in funding over the past 10–15 years, malaria continues to remain a widespread burden. The evidence of increasing resistance to anti-malarial drugs and insecticides is a growing concern. Hence, an efficacious and durable preventative vaccine for malaria is urgently needed. Vaccines are one of the most cost-effective tools and have successfully been used in the prevention and control of many diseases, however, the development of a vaccine for the Plasmodium parasite has proved difficult. Given the early success of whole sporozoite mosquito-bite delivered vaccination strategies, we know that a vaccine for malaria is an achievable goal, with sub-unit vaccines holding great promise as they are simple and cheap to both manufacture and deploy. However a major difficulty in development of sub-unit vaccines lies within choosing the appropriate antigenic target from the 5000 or so genes expressed by the parasite. Given the liver-stage of malaria represents a bottle-neck in the parasite’s life cycle, there is widespread agreement that a multi-component sub-unit malaria vaccine should preferably contain a liver-stage target. In this article we review progress in identifying and screening Plasmodium falciparum liver-stage targets for use in a malaria vaccine.