Sensitive detection of Plasmodium vivax malaria by the rotating-crystal magneto-optical method in Thailand

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

An in vitro assay to measure antibody-mediated inhibition of P. berghei sporozoite invasion against P. falciparum antigens.

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

Asymptomatic Plasmodium vivax infections induce robust IgG responses to multiple blood-stage proteins in a low-transmission region of western Thailand

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

Protective CD8+ T-cell immunity to human malaria induced by chimpanzee adenovirus-MVA immunisation.

Induction of antigen-specific CD8(+) T cells offers the prospect of immunization against many infectious diseases, but no subunit vaccine has induced CD8(+) T cells that correlate with efficacy in humans. Here we demonstrate that a replication-deficient chimpanzee adenovirus vector followed by a modified vaccinia virus Ankara booster induces exceptionally high frequency T-cell responses (median >2400 SFC/10(6) peripheral blood mononuclear cells) to the liver-stage Plasmodium falciparum malaria antigen ME-TRAP. It induces sterile protective efficacy against heterologous strain sporozoites in three vaccinees (3/14, 21%), and delays time to patency through substantial reduction of liver-stage parasite burden in five more (5/14, 36%), P=0.008 compared with controls. The frequency of monofunctional interferon-γ-producing CD8(+) T cells, but not antibodies, correlates with sterile protection and delay in time to patency (P(corrected)=0.005). Vaccine-induced CD8(+) T cells provide protection against human malaria, suggesting that a major limitation of previous vaccination approaches has been the insufficient magnitude of induced T cells

Using Serological Markers for the Surveillance of <i>Plasmodium vivax</i> Malaria: A Scoping Review

The utilisation of serological surveillance methods for malaria has the potential to identify individuals exposed to Plasmodium vivax, including asymptomatic carriers. However, the application of serosurveillance varies globally, including variations in methodology and transmission context. No systematic review exists describing the advantages and disadvantages of utilising serosurveillance in various settings. Collation and comparison of these results is a necessary first step to standardise and validate the use of serology for the surveillance of P. vivax in specific transmission contexts. A scoping review was performed of P. vivax serosurveillance applications globally. Ninety-four studies were found that met predefined inclusion and exclusion criteria. These studies were examined to determine the advantages and disadvantages of serosurveillance experienced in each study. If studies reported seroprevalence results, this information was also captured. Measurement of antibodies serves as a proxy by which individuals exposed to P. vivax may be indirectly identified, including those with asymptomatic infections, which may be missed by other technologies. Other thematic advantages identified included the ease and simplicity of serological assays compared to both microscopy and molecular diagnostics. Seroprevalence rates varied widely from 0–93%. Methodologies must be validated across various transmission contexts to ensure the applicability and comparability of results. Other thematic disadvantages identified included challenges with species cross-reactivity and determining changes in transmission patterns in both the short- and long-term. Serosurveillance requires further refinement to be fully realised as an actionable tool. Some work has begun in this area, but more is required

Host resistance to malaria : using mouse model to explore the host response

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.11 page(s

Characterization of splenocytes used in the <i>in vitro</i> assays.

<p>(A) Cellular immunogenicity of ChAd63-MVA <i>P</i>. <i>berghei</i> TRAP in C57BL/6 mice. Each data point represents splenocytes from two mice pooled together, with twelve pairs in total that were used in thirteen assays (one pair provided enough cells for two experiments). Cellular immunogenicity was assessed by ICS, after six hours stimulation with a pool of <i>P</i>. <i>berghei</i> TRAP peptides. Vector control mice were vaccinated with ChAd63-MVA luciferase and treated identically to the experimental mice. Results are expressed as the percentage of CD8⁺ cells, with median and individual data points shown. (B) Prior to addition of the splenocytes into the <i>in vitro</i> assays, samples were enriched for CD8⁺ cells. Results are expressed as the percentage of CD8⁺ cells out of total splenocytes, with both median and individual data points shown. (C) In each <i>in vitro</i> assay conducted, CD8⁺ enriched splenocytes from vector control vaccinated mice were included in the assay, along with wells containing sporozoites only, to act as controls. Results are expressed as the percentage infectivity, with the median shown for each experiment and error bars representing the interquartile range. (D) These controls allowed calculation of the background level of non-specific inhibition. Results are expressed as the percentage inhibition of splenocytes from vector control vaccinated mice compared to sporozoite only wells (no splenocytes), with median and individual data points shown for each experiment. In Exp1 and Exp2 sporozoite only wells were not included and hence the background inhibition could not be calculated.</p

Correlation of the percentage inhibition with the effector to target ratio.

<p>(A) Results are expressed as the percent inhibition compared to mock-vaccinated control wells (equal number of CD8⁺ enriched splenocytes from ChAd63-MVA luciferase vaccinated mice). Across all thirteen experiments there was no significant correlation between E:T ratio and inhibition (Spearman r = 0.35, p = 0.24), although a trend can be observed. Full circles represent those experiments that fit this trend, whilst empty circles represent those that did not. Experiments were then divided into data that fitted the E:T pattern (full circles) and those that did not (empty circles) and a graph (B) of the infectivity measured in wells containing only Hepa1-6 cells and sporozoites are shown. Statistical difference was assessed using the Mann Whitney test, **** p<0.0001. (C) Correlation of the E:T ratio with the percentage inhibition (n = 9 experiments), excluding the outliers. Spearman r = 0.82, p = 0.011.</p

The infectivity of the Hepa1-6 cell line.

<p>40 000 <i>P</i>. <i>berghei</i> GFP sporozoites were added per well with (n = 69) or without (n = 6) centrifugation. To determine whether dead or aborted sporozoites maintained expression of GFP, sporozoites were heat-killed for 20 minutes at 95°C prior to infection of Hepa1-6 cells (n = 6). 24 hours post-infection cells were harvested and run on a flow cytometer. (A) Representative example of the flow cytometry plots. (B) Data from multiple experiments was pooled and results are expressed as the percentage of GFP positive viable Hepa1-6 cells. The effect of centrifugation on infectivity was assessed using the Mann Whitney test, *** p = 0.0001.</p

Outline and characteristics of splenocytes used in the thirteen <i>in vitro</i> assays performed.

<p>^a PbTRAP-specific CD8⁺ cells: infected hepatocytes.</p><p>^b Sporozoite only wells were not included; number of infected hepatocytes was based on the median infectivity of 2.76%.</p><p>Outline and characteristics of splenocytes used in the thirteen <i>in vitro</i> assays performed.</p