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Comparing in vivo bioluminescence imaging and the Multi-Cruzi immunoassay platform to develop improved Chagas disease diagnostic procedures and biomarkers for monitoring parasitological cure [1]

['Amanda Fortes Francisco', 'Department Of Infection Biology', 'London School Of Hygiene', 'Tropical Medicine', 'Keppel Street', 'London', 'United Kingdom', 'Ursula Saade', 'Infynity Biomarkers', 'Lyon']

Date: 2022-11 

Abstract Background Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and is a serious public health problem throughout Latin America. With 6 million people infected, there is a major international effort to develop new drugs. In the chronic phase of the disease, the parasite burden is extremely low, infections are highly focal at a tissue/organ level, and bloodstream parasites are only intermittently detectable. As a result, clinical trials are constrained by difficulties associated with determining parasitological cure. Even highly sensitive PCR methodologies can be unreliable, with a tendency to produce “false-cure” readouts. Improved diagnostic techniques and biomarkers for cure are therefore an important medical need. Methodology/Principal findings Using an experimental mouse model, we have combined a multiplex assay system and highly sensitive bioluminescence imaging to evaluate serological procedures for diagnosis of T. cruzi infections and confirmation of parasitological cure. We identified a set of three antigens that in the context of the multiplex serology system, provide a rapid, reactive and highly accurate read-out of both acute and chronic T. cruzi infection. In addition, we describe specific antibody responses where down-regulation can be correlated with benznidazole-mediated parasite reduction and others where upregulation is associated with persistent infection. One specific antibody (IBAG39) highly correlated with the bioluminescence flux and represents a promising therapy monitoring biomarker in mice. Conclusions/Significance Robust, high-throughput methodologies for monitoring the efficacy of anti-T. cruzi drug treatment are urgently required. Using our experimental systems, we have identified markers of infection or parasite reduction that merit assessing in a clinical setting for the longitudinal monitoring of drug-treated patients.

Author summary Infections with the single-cell parasite Trypanosoma cruzi are life-long and cause Chagas disease. This can give rise to serious cardiac and/or digestive tract pathology. The current drugs are often non-curative, and patients frequently suffer severe side-effects. There is an international effort to develop improved therapeutic approaches. However, because T. cruzi persists at very low numbers in discrete tissue/organ foci, it is extremely difficult to unambiguously demonstrate parasitological cure. Even highly sensitive PCR methodologies require long-term follow-up to provide a curative diagnosis that is likely to be reliable. This is problematic for clinical trials and will complicate the roll-out of new drugs at a population level. Here, using experimental mouse models, we have exploited a combination of highly sensitive bioluminescence imaging and an antibody multiplex assay system to identify serological markers of infection and successful treatment. These biomarkers can now be adapted to provide a robust system for the diagnosis of Chagas disease and for confirming the parasitological cure of chronic infections.

Citation: Francisco AF, Saade U, Jayawardhana S, Pottel H, Scandale I, Chatelain E, et al. (2022) Comparing in vivo bioluminescence imaging and the Multi-Cruzi immunoassay platform to develop improved Chagas disease diagnostic procedures and biomarkers for monitoring parasitological cure. PLoS Negl Trop Dis 16(10): e0010827. https://doi.org/10.1371/journal.pntd.0010827 Editor: Igor C. Almeida, University of Texas at El Paso, UNITED STATES Received: May 17, 2022; Accepted: September 16, 2022; Published: October 3, 2022 Copyright: © 2022 Francisco et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the manuscript and its Supporting Information files. Funding: This work was supported by funding from the Drugs for Neglected Diseases initiative (DNDi) (https://dndi.org) to JMK and MZ. For this project, DNDi received financial support from the following donors: UK Aid, UK; Directorate-General for International Cooperation (DGIS), The Netherlands; Swiss Agency for Development and Cooperation, Switzerland; and for its overall mission from Médecins Sans Frontières (Doctors Without Borders), International. The donors had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Authors US, PL and MZ are all employed by INFYNITY BIOMARKERS. This does not alter their adherence to PLOS Neglected Tropical Diseases editorial policies and criteria. All other authors declare that they have no competing interests.

Introduction Chagas disease results from infection with the insect-transmitted, obligate intracellular parasite Trypanosoma cruzi, and is a major public health concern in many areas of Latin America [1, 2]. In humans, the initial acute stage is characterised by widespread distribution of T. cruzi in tissues and organs, and by a readily detectable parasitemia. The induction of a cellular immune response then results in a major reduction in the parasite load, and by 6–8 weeks post-infection, detection of bloodstream parasites becomes problematic. However, sterile clearance does not occur, and typically the infection transitions to a chronic life-long state [3]. In the acute stage, symptoms are usually mild and transient, although they can occasionally present as myocarditis or meningoencephalitis [4, 5]. 30–40% of infected people eventually develop chronic pathology, with cardiomyopathy and/or digestive megacolon syndromes being the most common outcomes [6, 7]. Chronic symptomatic disease can take decades to become apparent, and the lack of reliable predictive markers to identify those most at risk of disease progression has highlighted the need for new approaches in this area [8, 9]. There is strong evidence that the tissue damage associated with chronic Chagas disease is cumulative and dependent on parasite persistence [10–13]. This provides a rationale to underpin curative drug treatment as a strategy to block or alleviate the development of chronic pathology. The oral nitro-heterocyclic pro-drugs benznidazole and nifurtimox are the only chemotherapeutic treatments available for T. cruzi infections [14, 15]. Although treatments show efficacy in the chronic indeterminate stage of the disease, long treatment periods (~60 days) and frequent toxic side-effects impact negatively on patient compliance [16–18]. In addition, few of those in the acute or asymptomatic chronic stages are diagnosed, and the percentage of infected individuals offered anti-parasitic treatment, at the time-point when it would be most beneficial, is very small [19]. In response to the urgent need for more effective anti-T. cruzi therapeutics, there is now a global effort involving the academic, commercial, and not-for-profit sectors [20]. Furthermore, clinical trials aimed at optimising dosing regimens of the currently available drugs are on-going [21]. During the chronic stage, parasites are only intermittently detectable in the bloodstream and infection foci are rare and highly focal at a tissue/organ level [22]. As a result, unequivocal confirmation of parasitological cure is difficult to demonstrate. Even highly sensitive PCR methodologies require long-term follow-up to provide reliable diagnosis [16–17]. Serology-based approaches to assess parasitological cure have also proved to be problematic with seroconversion often requiring many years [23–24]. The identification of robust biomarkers of parasitological cure is therefore crucial to facilitate clinical trials and the subsequent roll-out of new drugs at a community level. The long-term nature and complexity of Chagas disease in humans has meant that predictive experimental models have played a key role in research [25]. One such example is the murine bioluminescence imaging system, based on infections with genetically modified T. cruzi that express a red-shifted luciferase, which facilitates highly sensitive longitudinal monitoring of parasite burden in a non-invasive manner [26–28]. This has become an important tool for drug testing and for studies on disease pathogenesis. Here, we have combined this pre-clinical model with an antibody multiplex assay system (Multi-Cruzi, InfYnity Biomarkers, Lyon, France) [29–31] as a means of developing improved serological procedures for diagnosis of Chagas disease and for confirming parasitological cure of chronic T. cruzi infections.

Methods Ethics statement Experiments were performed under UK Home Office project licenses PPL 70/8207 and P9AEE04E4, with consent of the LSHTM Animal Welfare and Ethical Review Board (AWERB). All protocols and procedures were conducted in accordance with the UK Animals (Scientific Procedures) Act 1986 (ASPA). Mice and parasites Experimental infections were carried out using female BALB/c mice, purchased from Charles River (UK). CB17-SCID mice were bred in-house. They were maintained in individually ventilated cages, under specific pathogen-free conditions, with a 12-hour light/dark cycle, and given food and water ad libitum. A T. cruzi CL Brener line (Discrete Typing Unit (DTU) 6), that constitutively expresses the red-shifted luciferase PpyRE9h [26], was used for infections. BALB/c mice (6–8 weeks old), were injected i.p. with 1x103 bloodstream trypomastigotes obtained from immunodeficient CB17-SCID mice, as outlined previously [27, 28]. Assessment of drug treatment and bioluminescence monitoring Chronically infected mice were dosed by oral gavage with benznidazole (Epichem Pty Ltd.) in an aqueous suspension vehicle containing 0.5% (w/v) hydroxypropyl methylcellulose (HPMC), 0.4% (w/v) Tween 80 in Milli-Q H 2 O. For in vivo imaging, mice were injected with 150 mg kg-1 d-luciferin i.p., and anaesthetized with 2.5% (v/v) gaseous isoflurane in oxygen [27, 28]. They were then placed in IVIS Lumina or Spectrum imaging systems (Caliper Life Science). Images were acquired 10–20 minutes after d-luciferin administration using Living Image 4.7.2. Exposure times were between 1 and 5 minutes, depending on the signal. To estimate parasite burden, whole body regions of interest were drawn using Living Image 4.5.5 to quantify bioluminescence expressed as total flux (photons/second; p/s). The detection threshold was established from uninfected mice. For ex vivo imaging, mice were injected i.p. with 150 mg kg−1 d-luciferin and then sacrificed by exsanguination under terminal anesthesia 5 minutes later [32]. They were perfused with 10 ml d-luciferin at 0.3 mg ml-1 in DPBS (Dulbecco’s phosphate-buffered saline) via the heart. Organs and tissues were excised, transferred to a dish, soaked in 0.3 mg d-luciferin ml-1 in DPBS, and then imaged as for the live mice. At various time points, blood samples (minimum 40 μl) were taken from tail veins into individual sterile tubes containing 10 μl 0.5 M EDTA. They were spun at 1,800 g for 10 minutes (4°C) and the serum fractions collected. Aliquots were stored at -20°C until shipment on dry ice. Multiplex Chagas assay The Multi-Cruzi immuno-assay used here is equivalent to the multiplex system described previously [29–31], adapted for mice serum samples. The technology allows multiple antigens to be combined in single wells of a 96-well microplate, using a printing process based on non-contact piezo electric impulsion of a defined volume of an antigenic solution. We used a non-contact volume dispensing system to print 16 T. cruzi antigens (Fig 1) at the bottom of each well at precise X-Y coordinates, under controlled humidity and temperature. Antigens, selected for proven immunogenic properties, were obtained synthetically and printed in duplicate [29, 30]. In addition, four Positive Control (PC) spots were printed in order to define the spatial orientation and validate the distribution of all the materials (mouse serum samples, conjugate and substrate) in the correct order. The ELISA assay was carried out as previously described [29] using the anti-mouse-IgG secondary antibody conjugated to horseradish peroxidase. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Antigens used in multiplex Chagas assay. (A) List of antigens. (B) Arrangement of antigens spotted in duplicate into the wells of 96-well microtiter plates. Four Positive Control (PC) spots (Methods) were printed to validate the test and give the spatial orientation of the matrix. https://doi.org/10.1371/journal.pntd.0010827.g001 Each plate was read and analysed using a specific microarray reader which acquires high-resolution digital images. An integrated software calculates the pixel intensity for each spot with the background noise subtracted. We calculated the mean value of the duplicated spots to get the net intensity of each antigen. The data were incorporated into a statistical analysis package and analysed accordingly. Study design Three cohorts of 15 BALB/c mice were infected with bioluminescent T. cruzi (Methods). Cohort size was selected to provide for the expected variability of the immune response, identified in preliminary experiments. When mice had reached the chronic stage (101 days post-infection), they were treated once daily with benznidazole at either 30 or 100 mg kg-1 for 5 days, dosing regimens that are generally non-curative or curative, respectively. These curative outcomes were based on mice being bioluminescence negative by both ex vivo and in vivo imaging following immunosuppressive treatment [33]. In the current experiment, mice were monitored by bioluminescence imaging, with blood samples taken at regular intervals: at days 101, 138, 182, 202, 259 and 300, relative to the day of infection (day 0). Statistical analysis Linear Mixed Model (LMM) analysis was used for the statistical analysis of the longitudinal data, since a mixed effects model has both random and fixed effects. In the current model, treatment group is a fixed effect, while time and mouse are random effects. Longitudinal data are described by the response variable (biomarker intensity), which was repeatedly measured at each group and time. The data prior to start of treatment were disregarded from the LMM analysis. There were no missing value observations. Response variables were the antigen reactivities of 16 biomarkers in the Multi-Cruzi assay and the log-transformed bioluminescence total flux. Independent variables were time (in days after infection) and treatment group (vehicle; benznidazole 30 mg kg-1; benznidazole 100 mg kg-1). Variance and co-variance were modelled as unstructured (UN). For each subgroup, and for each antigen or bioluminescence variable, slopes and intercepts were calculated and compared. SAS Proc Mixed was used for LMM analysis, from SAS 9.4 (SAS Institute Inc., Cary, NC, USA). The mixed effects model allows the average intercept and slope to be fitted as fixed effects, while taking account of differences between mice and time (random effects).

Discussion This study addresses one of the key challenges in Chagas disease control, which is the lack of serological tools and reliable biomarkers to monitor parasitological cure. The current strategy, for investigational settings, is to use PCR-based methods for assessing Chagas disease drug efficacy. However, because of the highly focal and low-level nature of chronic infections [22, 27], long-term follow-up is required [16, 17], and the potential for “false-cure” outcomes remains high. This presents major problems in clinical trials, and in future, will complicate the roll-out of new therapies. The recently developed multiplex serology assay system has shown potential as a means of monitoring T. cruzi persistence and identifying biomarkers that are predictive of successful treatment [29–31]. To further explore the utility of this approach, we applied the multiplex methodology to a highly sensitive experimental infection model, based on bioluminescence, where the limit of detection by ex vivo imaging is less than 20 parasites [22]. The aim was to determine if procedures such as this could be incorporated into the drug development pipeline, as an additional approach to identifying drug candidates that eliminate an infection. This experimental system also has the advantage of a standardized infection timeline and a method for designating cure that is more reliable than PCR-based methodologies [34]. During the acute stage, T. cruzi infection induces a strong polyclonal B cell/antibody response which can protect against virulent infection [35]. In the chronic stage however, the role of the humoral immune system is less certain [36]. The difficulties in addressing this reflect the diversity and complexity of the antibody response, even within an in-bred mouse population infected by a single T. cruzi clone (Fig 2). As shown, responses to some parasite antigens are rapid and almost universal (IBAG38, IBAG39, IBAG108 and IBAG257), whereas with others (eg. IBAG36, IBAG37 and IBAG101), induction is slow, but progressive. One of the most reactive antigens identified in the multiplex assay, IBAG257, corresponds to Tc24, a flagellar-localised Ca2+ binding protein that is highly conserved among different T. cruzi strains and is expressed throughout the development cycle [37, 38]. It has been widely tested as a vaccine candidate [39–41], and its use in serodiagnosis has been reported [42, 43]. Here, we found that the reactivity to this antigen using the multiplex assay was rapid, high and ubiquitous (n = 45) (Fig 2), but did not display discriminatory power to identify treated mice. A similar rapid and robust response was observed to IBAG39 (SAPA [44, 45]) and IBAG38, with all 45 mice in the current study (Fig 2), and all 25 mice in the pilot experiment, generating reactive antibodies. However, it was possible to discriminate between treated and non-treated mice on the basis of the reactivity towards these antigens. In response to 100 mg kg-1 benznidazole treatment, there was a slow but steady decline in the intensity of IBAG38 and IBAG39 antibody reactivity post-dosing, compared to a continued increase in the non-drug treated mice (Fig 3A and 3B), a profile that was highly correlated (for IBAG39) with the bioluminescence flux (Fig 5). The response was intermediate in the case of mice treated at 30 mg kg-1. Non-treated mice could also be discriminated by their response to IBAG36, IBAG37 and IBAG101. In the responding mice, the net intensity increased progressively from day 100 post-infection onwards, whereas there was no significant response in those mice that had been treated with 100 mg kg-1 (Fig 3D–3F). IBAG108 proved to be highly reactive, with a rapid and long-lasting response in >90% of the mice tested (Fig 2C), but showed no difference between treatment subgroups. Therefore, in the context of the multiplex system, IBAG38, IBAG39, IBAG108 and IBAG257 represent a highly sensitive and powerful combination for diagnosis of both acute and chronic T. cruzi infection, with IBAG39 having potential as a therapy-monitoring biomarker in mice. The multiplex system approach offers significant value for biomarker identification and validation due to its multi-parametric nature, with information collected simultaneously for several different antigens. Each antigen provides data on the development of reactivity and the probable antibody load in individual animals. The combination of independent parameters, enhances the analysis and provides a rapid, reliable and robust indication of signal decay linked to waning antibody levels. This approach, combined with recently developed prediction and dilution concepts based on mathematical models, is especially relevant for rapid evaluation of the dynamics of the antibody-mediated immune response [31, 46]. Treatment success is difficult to measure using the current criterion for parasitological cure because T. cruzi-specific antibodies persist for long periods after successful anti-parasitic treatment, and long-term follow-up is needed to monitor seroconversion. Detailed validation of the Multi-Cruzi immunoassay and selected clinically proven biomarkers is important as a demonstration of translational value—its usefulness for in vivo basic research and diagnostic applications for patients. One limitation of the current study concerns the restriction to the BALB/c mouse infection model and the T. cruzi CL Brener strain. Another could be the reported immune modulation associated with benznidazole treatment [47]. It will therefore be worthwhile to verify whether reduction of parasite load is also associated with a decrease of the specific serological markers when using other T. cruzi lines, mouse strains, and therapeutic agents. Finally, it would be useful to extend our study to other laboratory animals, such as naturally infected non-human primates, which can serve as a model for evaluating the effect of drug candidates on infection with this parasite [48], and for informing strategies applicable to a clinical context. In summary, the Multi-cruzi assay represents a highly promising research and discovery tool that can be used in combination with currently used diagnostic techniques (PCR and bioluminescence) to perform serological profiling studies and to monitor drug and possibly vaccine efficacy in preclinical models. Furthermore, its early adoption during the discovery process, and as a tool for clinical trial evaluation and regulatory approval, could help to drive acceptance of new treatments for Chagas disease.

Supporting information S1 Fig. Longitudinal monitoring of Trypanosoma cruzi-infected mice by in vivo bioluminescence imaging. 45 BALB/c mice were infected with bioluminescent T. cruzi (strain CL Brener) (Methods). At day 101 post-infection (dpi) (indicated by red arrow), the mice were treated with vehicle, 30 or 100 mg kg-1 benznidazole (BZ), once daily by the oral route for 5 days (n = 15 per group). They were monitored by in vivo imaging until the experimental end-point (Methods). For each group of 15 mice, ventral and dorsal images were captured in sets of 5, numbered left to right (#1–5, 6–10 and 11–15). All images use the same log10 scale heat-map with minimum and maximum radiance values as indicated. https://doi.org/10.1371/journal.pntd.0010827.s001 (PPTX) S2 Fig. Ex vivo imaging of benznidazole-treated mice at the experimental end-point. At 301 days post infection, mice that had been treated with 100 mg kg-1 benznidazole for 5 days (S1 Fig) were euthanized and subjected to ex vivo imaging (Methods). Bioluminescent foci (examples highlighted by white arrows) were detected in 4 mice, which were designated as non-cured. The organs and tissues are organized as shown in Fig 4. https://doi.org/10.1371/journal.pntd.0010827.s002 (PPTX) S3 Fig. Total whole body bioluminescence of mice at the experimental end-point. Mice (n = 15 per group) were imaged 300 days post-infection to establish whole body bioluminescence (sum of ventral and dorsal images) (Methods). (A) Control cohort administered with HPMC vehicle; (B) and (C) Cohorts treated with benznidazole (BZ) at 30 and 100 mg kg -1, respectively (Methods). See S1 Fig, to identify in vivo images of individual mice (numbering system described in the legend). In the 100 mg kg-1 cohort, the (+) symbol identifies mice shown to be non-cured by ex vivo imaging (S2 Fig). Dashed lines represent average background levels for naïve mice (n = 5). https://doi.org/10.1371/journal.pntd.0010827.s003 (PPTX)

Acknowledgments Recombinant Tc24-C4 protein, a Chagas disease therapeutic vaccine candidate was kindly provided by the team at Texas Children’s Hospital Center for Vaccine Development at Baylor College of Medicine (Texas, USA). The authors wish to thank Fanny Escudié for her coordination role of the in vivo studies.

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