Domestic Dog Infection with Trypanosoma cruzi from Northern and Southern Regions of Mexico.

Background: Chagas disease or American trypanosomiasis, caused by Trypanosoma cruzi and vectored by triatomines, affects millions of people worldwide. In endemic countries including Mexico, infections in domestic animals, such as dogs, may affect the risk of human disease when they serve as a source of infection to vectors that subsequently infect humans. Materials and Methods: We conducted a cross-sectional study of 296 dogs from two cities near the northern and southern borders of Mexico: Reynosa, Tamaulipas, and Tuxtla Gutierrez, Chiapas. Infection was measured based on testing of blood using T. cruzi quantitative PCR (qPCR) and up to three antibody detection assays. The StatPak immunochromatographic assay was used to screen samples and the indirect fluorescent antibody (IFA) and multiplex microsphere immunoassay (MIA) tests were used as secondary tests on all samples that screened positive and a subset of negatives. Serologic positivity was defined based on reactivity on at least two independent tests. Results: Of the 280 samples tested for parasite DNA, two (0.7%) were positive, one of which (0.4%) was confirmed as T. cruzi discrete typing unit TcIV. Overall, 72 (24.3%) samples were reactive for T. cruzi antibodies via StatPak of which 8 were also positive using MIA and 2 were also positive using IFA (including one of the PCR-positive dogs). Overall, nine dogs (3.4%) met study criteria of positivity based on either/both serology or PCR tests. Positive dogs were found in both regions of Mexico; five (2.7%) from Reynosa and four (3.6%) from Tuxtla Gutierrez. We found no association between infection status and state of origin, sex, age group, breed group, neighborhood, and whether other pets lived in the home. Conclusion: Our results re-emphasize dogs' utility as sentinels for T. cruzi in Mexico and underscore the need for improved veterinary diagnostic tests and parasite surveillance at the household level in endemic countries.

Positive clinical outcome using a modified dosing regimen of benznidazole in dogs at high risk for infection or acutely infected with Trypanosoma cruzi.

Trypanosoma cruzi infection in dogs can cause heart failure and sudden death with few treatment options available. A litter of 4 dogs living in a T cruzi endemic area were randomized to prophylaxis and nonprophylaxis groups as part of a study evaluating a modified benznidazole dosing regimen administered twice weekly to prevent T cruzi infection during a vector transmission season. The 2 dogs that received prophylaxis remained healthy without T cruzi infection or cardiac disease for >2 years. One dog that did not receive prophylaxis died unexpectedly with acute T cruzi-induced pancarditis, and the second dog tested positive for T cruzi and developed complex arrhythmias with markedly increased cardiac troponin I and improved with a higher benznidazole treatment dose. Although the small sample size precludes definitive conclusions, we describe the potential clinical benefit of prophylactic and early treatment with modified benznidazole dosing regimens for dogs with T cruzi infection.

Validation of a multiplex microsphere immunoassay for detection of antibodies to Trypanosoma cruzi in dogs.

The vector-borne protozoan parasite Trypanosoma cruzi causes Chagas disease in humans, dogs, and many other mammalian hosts. Canine Chagas disease is increasingly diagnosed in dogs of the southern United States where triatomine insect vectors occur, and there are limited veterinary testing options; only the indirect fluorescent antibody (IFA) test is offered at a single accredited diagnostic laboratory. We evaluated a multiplex microsphere immunoassay (MIA) for the detection of antibodies against T. cruzi in dogs and compared it with existing serologic methods to establish cutoff values and relative sensitivity and specificity. We tested 135 canine sera that had been characterized using the IFA and off-label use of 2 commercial rapid assays with our multiplex MIA against 12 antigens: 9 T. cruzi antigens, a negative control recombinant protein (green fluorescent protein, GFP), a Leishmania antigen, and a canine parvovirus antigen (used as an antibody control given near-ubiquitous parvoviral vaccination). The median fluorescence intensity (MFI) ratio between each T. cruzi antigen and GFP was calculated for every sample. Samples with an antigen:GFP MFI ratio > 4 SDs above the mean of 25 known-negative sera were considered positive to that antigen. Samples testing positive to ≥ 2 antigens were considered positive for T. cruzi antibodies. Compared to the IFA, our multiplex MIA had a relative sensitivity of 100% and specificity of 97.0%. Given its precision, high-throughput format, potential for automation, and lack of subjective interpretation, our multiplex MIA should be considered a valid and improved assay for T. cruzi antibodies in dogs.

cGAS-STING Pathway Activation during Trypanosoma cruzi Infection Leads to Tissue-Dependent Parasite Control.

Host cell invasion by Trypanosoma cruzi is a markedly silent process, with limited host transcriptional changes indicative of innate immune recognition, except for a modest type I IFN (IFN-I) response. In this study, we show that T. cruzi-induced IFN-ß production was nearly abolished in primary murine cGAS-/- or stimulator of IFN genes (STING)-deficient (STINGGt) macrophages and fibroblasts. T. cruzi infection did not impact the ability of IFN-regulatory factor reporter macrophages to respond to classical cGAS-STING agonists, indicating that the limited IFN-ß induction is not due to active parasite suppression. cGAS-/-, STINGGt, and IFN-α/ß receptor-/- (IFNAR-/-) macrophages infected with T. cruzi yielded significantly higher numbers of amastigotes compared with wild-type macrophages; however, the impact of the STING pathway during infection in vivo is more complex. Despite an initial increase in parasite growth, STINGGt and IFNAR-/- mice ultimately had lower parasite burden in footpads as compared with wild-type mice, demonstrating a role for IFN-I expression in potentiating parasite growth at the infection site. STING pathway activation had little impact on parasite levels in the skeletal muscle; however, in the heart, cGAS-/- and STINGGt mice, but not IFNAR-/- mice, accumulated higher acute parasite loads, suggesting a protective role of STING sensing of T. cruzi in this organ that was independent of IFN-I. Together, these results demonstrate that host cGAS-STING senses T. cruzi infection, enhancing parasite growth at the site of entry, and contributes to acute-phase parasite restriction in the heart, a major site of tissue damage in chronic T. cruzi infection.

DIAGNOSIS AND TREATMENT OF A NATURAL INFECTION WITH TRYPANOSOMA CRUZI (CHAGAS DISEASE) IN A SYMPTOMATIC DE BRAZZA'S MONKEY (CERCOPITHECUS NEGLECTUS) IN ALABAMA.

Trypanosoma cruzi, the causative agent of Chagas disease, is a zoonotic, vector-borne, protozoan hemoflagellate with a wide host range. An 11-yr-old, captive-bred male De Brazza's monkey (Cercopithecus neglecus) presented with weight loss despite normal appetite. Examination revealed hypoglycemia, nonregenerative anemia, and many trypanosomes on a blood smear. A whole blood sample was PCR-positive for T. cruzi discrete typing unit TcIV and the monkey seroconverted using two different methods. The monkey was treated with the standard human dose of benznidazole twice daily for 60 d; however, blood obtained over the next 1.5 yr posttreatment remained PCR-positive for T. cruzi. A second course of benznidazole at a higher dose but lower frequency for 26 wk was required for the monkey to convert to sustained PCR-negative status. The monkey recovered with no apparent lasting effects.

Frequency Variation and Dose Modification of Benznidazole Administration for the Treatment of Trypanosoma cruzi Infection in Mice, Dogs, and Nonhuman Primates.

Trypanosoma cruzi naturally infects a broad range of mammalian species and frequently results in the pathology that has been most extensively characterized in human Chagas disease. Currently employed treatment regimens fail to achieve parasitological cure of T. cruzi infection in the majority of cases. In this study, we have extended our previous investigations of more effective, higher dose, intermittent administration protocols using the FDA-approved drug benznidazole (BNZ), in experimentally infected mice and in naturally infected dogs and nonhuman primates (NHP). Collectively, these studies demonstrate that twice-weekly administration of BNZ for more than 4 months at doses that are ~2.5-fold that of previously used daily dosing protocols, provided the best chance to obtain parasitological cure. Dosing less frequently or for shorter time periods was less dependable in all species. Prior treatment using an ineffective dosing regimen in NHPs did not prevent the attainment of parasitological cure with an intensified BNZ dosing protocol. Furthermore, parasites isolated after a failed BNZ treatment showed nearly identical susceptibility to BNZ as those obtained prior to treatment, confirming the low risk of induction of drug resistance with BNZ and the ability to adjust the treatment protocol when an initial regimen fails. These results provide guidance for the use of BNZ as an effective treatment for T. cruzi infection and encourage its wider use, minimally in high value dogs and at-risk NHP, but also potentially in humans, until better options are available.

Effective drug discovery in Chagas disease.

The Chagas field has gone >50 years without tangible progress toward new therapies. My colleagues and I have recently reported on a benzoxaborole compound that achieves consistent parasitological cure in experimentally infected mice and in naturally infected non-human primates (NHPs). While these results do not assure success in human clinical trials, they significantly de-risk this process and form a strong justification for such trials. Highly effective drug discovery depends on a solid understanding of host and parasite biology and excellent knowledge in designing and validating chemical entities. This opinion piece seeks to provide perspectives on the process that led to the discovery of AN15368, with the hope that this will facilitate the discovery of additional clinical candidates for Chagas disease.

Frequency variation and dose modification of benznidazole administration for the treatment of Trypanosoma cruzi infection in mice, dogs and non-human primates.

Trypanosoma cruzi naturally infects a broad range of mammalian species and frequently results in the pathology that has been most extensively characterized in human Chagas disease. Currently employed treatment regimens fail to achieve parasitological cure of T. cruzi infection in the majority of cases. In this study, we have extended our previous investigations of more effective, higher dose, intermittent administration protocols using the FDA-approved drug benznidazole (BNZ), in experimentally infected mice and in naturally infected dogs and non-human primates (NHP). Collectively these studies demonstrate that twice-weekly administration of BNZ for more than 4 months at doses that are ∻2.5-fold that of previously used daily dosing protocols, provided the best chance to obtain parasitological cure. Dosing less frequently or for shorter time periods was less dependable in all species. Prior treatment using an ineffective dosing regimen in NHPs did not prevent the attainment of parasitological cure with an intensified BNZ dosing protocol. Furthermore, parasites isolated after a failed BNZ treatment showed nearly identical susceptibility to BNZ as those obtained prior to treatment, confirming the low risk of induction of drug resistance with BNZ and the ability to adjust the treatment protocol when an initial regimen fails. These results provide guidance for the use of BNZ as an effective treatment for T. cruzi infection and encourage its wider use, minimally in high value dogs and at-risk NHP, but also potentially in humans, until better options are available.

Delayed Activation of T Cells at the Site of Infection Facilitates the Establishment of Trypanosoma cruzi in Both Naive and Immune Hosts.

Although parasite entry through breaks in the skin or mucosa is one of the main routes of natural transmission of Trypanosoma cruzi, little is known about the host cell types initially invaded nor the ability of those host cells to initiate immune responses at the site of infection. To gain insights into these early events, we studied the fate of fluorescently tagged T. cruzi delivered subcutaneously in mouse footpads or ears. We demonstrate that the majority of parasites introduced into the skin initially proliferate there until 8 to 10 days postinfection, when the parasite load decreases. This decline in parasite numbers is dependent on the presence of an intact T cell compartment and on the ability of hosts to produce gamma interferon (IFN-γ). Many of the parasite-containing cells at the initial infection site display a macrophage/monocyte phenotype but with low expression of activation markers, suggesting that these cells provide an early niche for T. cruzi proliferation, rather than being active in parasite control. It is only after the first round of T. cruzi replication and release from host cells that signs of immune activation and control of parasites become apparent. The delay in the activation and failure to rapidly control parasite replication are observed even when T. cruzi-primed T cells are present, such as in chronically infected mice. This failure of a primed immune system to recognize and react prior to extensive parasite expansion at the infection site likely poses a significant challenge for the development of vaccines aiming to prevent T. cruzi infection. IMPORTANCE Trypanosoma cruzi, the parasite causing Chagas disease, usually infects through the mucosa or breaks in the skin, but little is known about the parasite's fate at the site of entry or the early events involving immune control there. Here, we track the local proliferation and subsequent dissemination of fluorescently tagged T. cruzi and the initial immune response at the point of entry. We show that T. cruzi preferentially infects innate immune cells in the skin and that the stimulation of an adaptive T cell response does not occur until after the release of parasites from this first round of infected host cells. This first immunologically "silent" proliferation occurs even in the presence of a strong immune T cell memory generated by previous infection. This capacity of T. cruzi to establish infections while avoiding initial immune recognition has important implications for the potential to develop vaccines to prevent T. cruzi infection.

Epitopes in the Glycosylphosphatidylinositol Attachment Signal Peptide of Trypanosoma cruzi Mucin Proteins Generate Robust but Delayed and Nonprotective CD8+ T Cell Responses.

Infection with the protozoan parasite Trypanosoma cruzi elicits substantial CD8+ T cell responses that disproportionately target epitopes encoded in the large trans-sialidase (TS) gene family. Within the C57BL/6 infection model, a significant proportion (30-40%) of the T. cruzi-specific CD8+ T cell response targets two immunodominant TS epitopes, TSKb18 and TSKb20. However, both TS-specific CD8+ T cell responses are dispensable for immune control, and TS-based vaccines have no demonstrable impact on parasite persistence, a determinant of disease. Besides TS, the specificity and protective capacity of CD8+ T cells that mediate immune control of T. cruzi infection are unknown. With the goal of identifying alternative CD8+ T cell targets, we designed and screened a representative set of genome-wide, in silico-predicted epitopes. Our screen identified a previously uncharacterized, to our knowledge, T cell epitope MUCKb25, found within mucin family proteins, the third most expanded large gene family in T. cruzi. The MUCKb25-specific response was characterized by delayed kinetics, relative to TS-specific responses, and extensive cross-reactivity with a large number of endogenous epitope variants. Similar to TS-specific responses, the MUCKb25 response was dispensable for control of the infection, and vaccination to generate MUCK-specific CD8+ T cells failed to confer protection. The lack of protection by MUCK vaccination was partly attributed to the fact that MUCKb25-specific T cells exhibit limited recognition of T. cruzi-infected host cells. Overall, these results indicate that the CD8+ T cell compartment in many T. cruzi-infected mice is occupied by cells with minimal apparent effector potential.

Avoiding Clinical Trial Failures in Neglected Tropical Diseases: The Example of Chagas Disease.

Human clinical trials are expensive, and when they fail, they create the impression that a problem is intractable, thus depressing interest in future attempts. For neglected tropical diseases, where there are likely limited numbers of "shots on goal," such failures need to be assiduously avoided. Chagas disease drug discovery efforts have experienced more than its share of human clinical trial failures. Here are some guidelines, many specific for Chagas, but some that might also have application for other neglected tropical diseases. Chagas disease has major challenges (eg, the lack of a definitive test of cure) but also has outstanding advantages, among these the unmatched multi-species natural infection systems that can be exploited to de-risk compounds before human trials. Fully utilizing these advantages while frankly acknowledging and addressing the challenges should bring better options to patients, sooner.

Prophylactic low-dose, bi-weekly benznidazole treatment fails to prevent Trypanosoma cruzi infection in dogs under intense transmission pressure.

Trypanosoma cruzi naturally infects a wide variety of wild and domesticated mammals, in addition to humans. Depending on the infection dose and other factors, the acute infection can be life-threatening, and in all cases, the risk of chagasic heart disease is high in persistently infected hosts. Domestic, working, and semi-feral dogs in the Americas are at significant risk of T. cruzi infection and in certain settings in the southern United States, the risk of new infections can exceed 30% per year, even with the use of vector control protocols. In this study, we explored whether intermittent low-dose treatment with the trypanocidal compound benznidazole (BNZ) during the transmission season, could alter the number of new infections in dogs in an area of known, intense transmission pressure. Preliminary studies in mice suggested that twice-weekly administration of BNZ could prevent or truncate infections when parasites were delivered at the mid-point between BNZ doses. Pre-transmission season screening of 126 dogs identified 53 dogs (42.1%) as T. cruzi infection positive, based upon blood PCR and Luminex-based serology. Serial monitoring of the 67 uninfected dogs during the high transmission season (May to October) revealed 15 (22.4%) new infections, 6 in the untreated control group and 9 in the group receiving BNZ prophylaxis, indicating no impact of this prophylaxis regimen on the incidence of new infections. Although these studies suggest that rigorously timed and more potent dosing regimen may be needed to achieve an immediate benefit of prophylaxis, additional studies would be needed to determine if drug prophylaxis reduced disease severity despite this failure to prevent new infections.

Discovery of an orally active benzoxaborole prodrug effective in the treatment of Chagas disease in non-human primates.

Trypanosoma cruzi, the agent of Chagas disease, probably infects tens of millions of people, primarily in Latin America, causing morbidity and mortality. The options for treatment and prevention of Chagas disease are limited and underutilized. Here we describe the discovery of a series of benzoxaborole compounds with nanomolar activity against extra- and intracellular stages of T. cruzi. Leveraging both ongoing drug discovery efforts in related kinetoplastids, and the exceptional models for rapid drug screening and optimization in T. cruzi, we have identified the prodrug AN15368 that is activated by parasite carboxypeptidases to yield a compound that targets the messenger RNA processing pathway in T. cruzi. AN15368 was found to be active in vitro and in vivo against a range of genetically distinct T. cruzi lineages and was uniformly curative in non-human primates (NHPs) with long-term naturally acquired infections. Treatment in NHPs also revealed no detectable acute toxicity or long-term health or reproductive impact. Thus, AN15368 is an extensively validated and apparently safe, clinically ready candidate with promising potential for prevention and treatment of Chagas disease.

Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs.

Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi (T. cruzi), is an obligate intracellular parasite with a diverse biology that infects several mammalian species, including humans, causing cardiac and digestive pathologies. Reliable detection of T. cruzi in vivo infections has long been needed to understand Chagas disease's complex biology and accurately evaluate the outcome of treatment regimens. The current protocol demonstrates an integrated pipeline for automated quantification of T. cruzi-infected cells in 3D-reconstructed, cleared organs. Light-sheet fluorescent microscopy allows for accurately visualizing and quantifying of actively proliferating and dormant T. cruzi parasites and immune effector cells in whole organs or tissues. Also, the CUBIC-HistoVision pipeline to obtain uniform labeling of cleared organs with antibodies and nuclear stains was successfully adopted. Tissue clearing coupled with 3D immunostaining provides an unbiased approach to comprehensively evaluate drug treatment protocols, improve the understanding of the cellular organization of T. cruzi-infected tissues, and is expected to advance discoveries related to anti-T. cruzi immune responses, tissue damage, and repair in Chagas disease.

Application of SARS-CoV-2 Serology to Address Public Health Priorities.

Background: Antibodies against SARS-CoV-2 can be detected by various testing platforms, but a detailed understanding of assay performance is critical. Methods: We developed and validated a simple enzyme-linked immunosorbent assay (ELISA) to detect IgG binding to the receptor-binding domain (RBD) of SARS-CoV-2, which was then applied for surveillance. ELISA results were compared to a set of complimentary serologic assays using a large panel of clinical research samples. Results: The RBD ELISA exhibited robust performance in ROC curve analysis (AUC> 0.99; Se = 89%, Sp = 99.3%). Antibodies were detected in 23/353 (6.5%) healthcare workers, 6/9 RT-PCR-confirmed mild COVID-19 cases, and 0/30 non-COVID-19 cases from an ambulatory site. RBD ELISA showed a positive correlation with neutralizing activity (p = <0.0001, R2 = 0.26). Conclusions: We applied a validated SARS-CoV-2-specific IgG ELISA in multiple contexts and performed orthogonal testing on samples. This study demonstrates the utility of a simple serologic assay for detecting prior SARS-CoV-2 infection, particularly as a tool for efficiently testing large numbers of samples as in population surveillance. Our work also highlights that precise understanding of SARS-CoV-2 infection and immunity at the individual level, particularly with wide availability of vaccination, may be improved by orthogonal testing and/or more complex assays such as multiplex bead assays.

Immune exhaustion in chronic Chagas disease: Pro-inflammatory and immunomodulatory action of IL-27 in vitro.

In chronic Chagas disease, Trypanosoma cruzi-specific T-cell function decreases over time, and alterations in the homeostatic IL-7/IL-7R axis are evident, consistent with a process of immune exhaustion. IL-27 is an important immunoregulatory cytokine that shares T-cell signaling with IL-7 and other cytokines of the IL-12 family and might be involved in the transcriptional regulation of T-cell function. Here, we evaluated the expression and function of IL-27R in antigen-experienced T cells from subjects with chronic Chagas disease and assessed whether in vitro treatment with IL-27 and IL-7 might improve T. cruzi-specific polyfunctional T-cell responses. In vitro exposure of PBMCs to T. cruzi induced a downregulation of IL-27R in CD4+ T cells and an upregulation in CD8+ T cells in subjects without heart disease, while IL-27R expression remained unaltered in subjects with more severe clinical stages. The modulation of IL-27R was associated with functional signaling through STAT3 and STAT5 and induction of the downstream genes TBX21, EOMES and CXCL9 in response to IL-27. In vitro treatment of PBMCs with IL-27 and IL-7 improved monofunctional and polyfunctional Th1 responses, accompanied by the induction of IL-10 and Bcl-2 expression in subjects without heart disease but did not improve those in subjects with cardiomyopathy. Our findings support the process of desensitization of the IL-27/IL-27R pathway along with disease severity and that the pro-inflammatory and immunomodulatory mechanisms of IL-27 might be interconnected.

High variation in immune responses and parasite phenotypes in naturally acquired Trypanosoma cruzi infection in a captive non-human primate breeding colony in Texas, USA.

Trypanosoma cruzi, the causative agent of human Chagas disease, is endemic to the southern region of the United States where it routinely infects many host species. The indoor/outdoor housing configuration used in many non-human primate research and breeding facilities in the southern of the USA provides the opportunity for infection by T. cruzi and thus provides source material for in-depth investigation of host and parasite dynamics in a natural host species under highly controlled and restricted conditions. For cynomolgus macaques housed at such a facility, we used a combination of serial blood quantitative PCR (qPCR) and hemoculture to confirm infection in >92% of seropositive animals, although each method alone failed to detect infection in >20% of cases. Parasite isolates obtained from 43 of the 64 seropositive macaques were of 2 broad genetic types (discrete typing units, (DTU's) I and IV); both within and between these DTU groupings, isolates displayed a wide variation in growth characteristics and virulence, elicited host immune responses, and susceptibility to drug treatment in a mouse model. Likewise, the macaques displayed a diversity in T cell and antibody response profiles that rarely correlated with parasite DTU type, minimum length of infection, or age of the primate. This study reveals the complexity of infection dynamics, parasite phenotypes, and immune response patterns that can occur in a primate group, despite being housed in a uniform environment at a single location, and the limited time period over which the T. cruzi infections were established.

Reduced Trypanosoma cruzi-specific humoral response and enhanced T cell immunity after treatment interruption with benznidazole in chronic Chagas disease.

BACKGROUND: Interruption of benznidazole therapy due to the appearance of adverse effects, which is presumed to lead to treatment failure, is a major drawback in the treatment of chronic Chagas disease. METHODS: Trypanosoma cruzi-specific humoral and T cell responses, T cell phenotype and parasite load were measured to compare the outcome in 33 subjects with chronic Chagas disease treated with an incomplete benznidazole regimen and 58 subjects treated with the complete regimen, during a median follow-up period of 48 months. RESULTS: Both treatment regimens induced a reduction in the T. cruzi-specific antibody levels and similar rates of treatment failure when evaluated using quantitative PCR. Regardless of the regimen, polyfunctional CD4+ T cells increased in the subjects, with successful treatment outcome defined as a decrease of T. cruzi-specific antibodies. Regardless of the serological outcome, naive and central memory T cells increased after both regimens. A decrease in CD4+ HLA-DR+ T cells was associated with successful treatment in both regimens. The cytokine profiles of subjects with successful treatment showed fewer inflammatory mediators than those of the untreated T. cruzi-infected subjects. High levels of T cells expressing IL-7 receptor and low levels of CD8+ T cells expressing the programmed cell death protein 1 at baseline were associated with successful treatment following benznidazole interruption. CONCLUSIONS: These findings challenge the notion that treatment failure is the sole potential outcome of an incomplete benznidazole regimen and support the need for further assessment of the treatment protocols for chronic Chagas disease.

A flexible, pan-species, multi-antigen platform for the detection and monitoring of SARS-CoV-2-specific antibody responses.

The SARS-CoV-2 pandemic and the vaccination effort that is ongoing has created an unmet need for accessible, affordable, flexible and precise platforms for monitoring the induction, specificity and maintenance of virus-specific immune responses. Herein we validate a multiplex (Luminex-based) assay capable of detecting SARS-CoV-2-specific antibodies irrespective of host species, antibody isotype, and specimen type (e.g. plasma, serum, saliva or blood spots). The well-established precision of Luminex-based assays provides the ability to follow changes in antibody levels over time to many antigens, including multiple permutations of the most common SARS-CoV-2 antigens. This platform can easily measure antibodies known to correlate with neutralization activity as well as multiple non-SARS-CoV-2 antigens such as vaccines (e.g. Tetanus toxoid) or those from frequently encountered agents (influenza), which serve as stable reference points for quantifying the changing SARS-specific responses. All of the antigens utilized in our study can be made in-house, many in E. coli using readily available plasmids. Commercially sourced antigens may also be incorporated and newly available antigen variants can be rapidly produced and integrated, making the platform adaptable to the evolving viral strains in this pandemic.

Strain-specific genome evolution in Trypanosoma cruzi, the agent of Chagas disease.

The protozoan Trypanosoma cruzi almost invariably establishes life-long infections in humans and other mammals, despite the development of potent host immune responses that constrain parasite numbers. The consistent, decades-long persistence of T. cruzi in human hosts arises at least in part from the remarkable level of genetic diversity in multiple families of genes encoding the primary target antigens of anti-parasite immune responses. However, the highly repetitive nature of the genome-largely a result of these same extensive families of genes-have prevented a full understanding of the extent of gene diversity and its maintenance in T. cruzi. In this study, we have combined long-read sequencing and proximity ligation mapping to generate very high-quality assemblies of two T. cruzi strains representing the apparent ancestral lineages of the species. These assemblies reveal not only the full repertoire of the members of large gene families in the two strains, demonstrating extreme diversity within and between isolates, but also provide evidence of the processes that generate and maintain that diversity, including extensive gene amplification, dispersion of copies throughout the genome and diversification via recombination and in situ mutations. Gene amplification events also yield significant copy number variations in a substantial number of genes presumably not required for or involved in immune evasion, thus forming a second level of strain-dependent variation in this species. The extreme genome flexibility evident in T. cruzi also appears to create unique challenges with respect to preserving core genome functions and gene expression that sets this species apart from related kinetoplastids.