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Trypanosoma cruzi is a protozoan parasite that causes Chagas disease. Globally 6 to 7 million people are infected by this parasite of which 20-30% will progress to develop Chronic Chagasic Cardiomyopathy (CCC). Despite its high disease burden, no clinically approved vaccine exists for the prevention or treatment of CCC. Developing vaccines that can stimulate T. cruzi-specific CD8+ cytotoxic T cells and eliminate infected cells requires targeting parasitic antigens presented on major histocompatibility complex-I (MHC-I) molecules. We utilized mass spectrometry-based immunopeptidomics to investigate which parasitic peptides are displayed on MHC-I of T. cruzi infected cells. Through duplicate experiments, we identified an array of unique peptides that could be traced back to 17 distinct T. cruzi proteins. Notably, six peptides were derived from Tcj2, a trypanosome chaperone protein and member of the DnaJ (heat shock protein 40) family, showcasing its potential as a viable candidate vaccine antigen with cytotoxic T cell inducing capacity. Upon testing Tcj2 as an mRNA vaccine candidate in mice, we observed a strong memory cytotoxic CD8+ T cell response along with a Th1-skewed humoral antibody response. In vitro co-cultures of T. cruzi infected cells with splenocytes of Tcj2-immunized mice restricted the replication of T. cruzi, demonstrating the protective potential of Tcj2 as a vaccine target. Moreover, antisera from Tcj2-vaccinated mice displayed no cross-reactivity with DnaJ in lysates from mouse and human indicating a decreased likelihood of triggering autoimmune reactions. Our findings highlight how immunopeptidomics can identify new vaccine targets for Chagas disease, with Tcj2 emerging as a promising new mRNA vaccine candidate.

Abstract The development of broad-spectrum coronavirus vaccines is essential to prepare for future respiratory virus pandemics. We demonstrated broad neutralization by a trivalent subunit vaccine, formulating the receptor-binding domains of SARS-CoV, MERS-CoV, and SARS-CoV-2 XBB.1.5 with Alum and CpG55.2. Vaccinated mice produced cross-neutralizing antibodies against all three human Betacoronaviruses and others currently exclusive to bats, indicating the epitope preservation of the individual antigens during co-formulation and the potential for epitope broadening.

Introduction The development of a yeast-expressed recombinant protein-based vaccine technology co-developed with LMIC vaccine producers and suitable as a COVID-19 vaccine for global access is described. The proof-of-concept for developing a SARS-CoV-2 spike protein receptor-binding domain (RBD) antigen as a yeast-derived recombinant protein vaccine technology is described. Areas Covered Genetic Engineering: The strategy is presented for the design and genetic modification used during cloning and expression in the yeast system. Process and Assay Development: A summary is presented of how a scalable, reproducible, and robust production process for the recombinant protein COVID-19 vaccine antigen was developed. Formulation and Pre-clinical Strategy: We report on the pre-clinical and formulation strategy used for the proof-of-concept evaluation of the SARS-CoV-2 RBD vaccine antigen. Technology Transfer and Partnerships: The process used for the technology transfer and co-development with LMIC vaccine producers is described. Clinical Development and Delivery: The approach used by LMIC developers to establish the industrial process, clinical development, and deployment is described. Expert Opinion Highlighted is an alternative model for developing new vaccines for emerging infectious diseases of pandemic importance starting with an academic institution directly transferring their technology to LMIC vaccine producers without the involvement of multinational pharma companies.

Over the past three years, new SARS-CoV-2 variants have continuously emerged, evolving to a point where an immune response against the original vaccine no longer provided optimal protection against these new strains. During this time, high-throughput neutralization assays based on pseudoviruses have become a valuable tool for assessing the efficacy of new vaccines, screening updated vaccine candidates against emerging variants, and testing the efficacy of new therapeutics such as monoclonal antibodies. Lentiviral vectors derived from HIV-1 are popular for developing pseudo and chimeric viruses due to their ease of use, stability, and long-term transgene expression. However, the HIV-based platform has lower transduction rates for pseudotyping coronavirus spike proteins than other pseudovirus platforms, necessitating more optimized methods. As the SARS-CoV-2 virus evolved, we produced over 18 variants of the spike protein for pseudotyping with an HIV-based vector, optimizing experimental parameters for their production and transduction. In this article, we present key parameters that were assessed to improve such technology, including (a) the timing and method of collection of pseudovirus supernatant; (b) the timing of host cell transduction; (c) cell culture media replenishment after pseudovirus adsorption; and (d) the centrifugation (spinoculation) parameters of the host cell+ pseudovirus mix, towards improved transduction. Additionally, we found that, for some pseudoviruses, the addition of a cationic polymer (polybrene) to the culture medium improved the transduction process. These findings were applicable across variant spike pseudoviruses that include not only SARS-CoV-2 variants, but also SARS, MERS, Alpha Coronavirus (NL-63), and bat-like coronaviruses. In summary, we present improvements in transduction efficiency, which can broaden the dynamic range of the pseudovirus titration and neutralization assays.

IntroductionHookworms are parasitic helminths that secrete a variety of proteins that induce anti-inflammatory immune responses, stimulating increased CD4+Foxp3+ regulatory T cells and IL-10 production. Hookworm-derived recombinant proteins AIP-1 and AIP-2 have been shown to reduce inflammation in mouse models of inflammatory bowel disease and inflammatory airway disease by inducing CD4+Foxp3+ cells and IL-10 production. In contrast, chronic infection with the protozoal parasite Trypanosoma cruzi, the causative agent of Chagas disease, leads to chronic inflammation in tissues. Persistence of the parasites in tissues drives chronic low-grade inflammation, with increased infiltration of inflammatory cells into the heart, accompanied by increased production of inflammatory cytokines. There are no current antiparasitic drugs that effectively reduce or prevent chronic myocarditis caused by the onset of Chagas disease, thus new therapies are urgently needed. Therefore, the impact of AIP-1 and AIP-2 on myocarditis was investigated in a mouse model of chronic T. cruzi infection. MethodsFemale BALB/c mice infected with bioluminescent T. cruzi H1 strain trypomastigotes for 70 days were treated once daily for 7 days with 1mg/kg AIP-1 or AIP-2 protein by intraperitoneal injection. Control mice were left untreated or treated once daily for 14 days with 25mg/kg aspirin in drinking water. At 84 days of infection, splenocytes, cardiac tissue and serum were collected for evaluation. ResultsTreatment with both AIP-1 and AIP-2 proteins significantly reduced cardiac cellular infiltration, and reduced cardiac levels of IFNγ, IL-6 and IL-2. AIP-2 treatment reduced cardiac expression of COX-2. Further, while incubation with AIP-1 and AIP-2 proteins did not induce a significant upregulation of an immunoregulatory phenotype in dendritic cells (DC), there was a modest upregulation of CD11c+CD11b+MHCII+SIRPα+ expression, suggesting a regulatory phenotype. Ex-vivo stimulation of splenocytes from the treatment groups with AIP-1 loaded DC induced reduced levels of cytotoxic and pro-inflammatory T cells, stimulation with AIP-2 loaded DC specifically induced enhanced levels of CD4+CD25+Foxp3+ regulatory T cells among treatment groups. DiscussionAll in vivo and in vitro results demonstrate that hookworm-derived AIP-1 and AIP-2 proteins reduce T. cruzi induced cardiac inflammation, possibly through multiple anti-inflammatory mechanisms.

Tc24 is a Trypanosoma cruzi-derived flagellar protein that, when formulated with a TLR-4 agonist adjuvant, induces a balanced immune response in mice, elevating IgG2a antibody titers and IFN-γ levels. Furthermore, vaccination with the recombinant Tc24 protein can reduce parasite levels and improve survival during acute infection. Although some mRNA vaccines have been proven to elicit a stronger immune response than some protein vaccines, they have not been used against T. cruzi. This work evaluates the immunogenicity of a heterologous prime/boost vaccination regimen using protein and mRNA-based Tc24 vaccines. Mice (C57BL/6) were vaccinated twice subcutaneously, three weeks apart, with either the Tc24-C4 protein + glucopyranosyl A (GLA)-squalene emulsion, Tc24 mRNA Lipid Nanoparticles, or with heterologous protein/mRNA or mRNA/protein combinations, respectively. Two weeks after the last vaccination, mice were euthanized, spleens were collected to measure antigen-specific T-cell responses, and sera were collected to evaluate IgG titers and isotypes. Heterologous presentation of the Tc24 antigen generated antigen-specific polyfunctional CD8+ T cells, a balanced Th1/Th2/Th17 cytokine profile, and a balanced humoral response with increased serum IgG, IgG1 and IgG2c antibody responses. We conclude that heterologous vaccination using Tc24 mRNA to prime and Tc24-C4 protein to boost induces a broad and robust antigen-specific immune response that was equivalent or superior to two doses of a homologous protein vaccine, the homologous mRNA vaccine and the heterologous Tc24-C4 Protein/mRNA vaccine.

(1) Background: We previously reported the development of a recombinant protein SARS-CoV-2 vaccine, consisting of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, adjuvanted with aluminum hydroxide (alum) and CpG oligonucleotides. In mice and non-human primates, our wild-type (WT) RBD vaccine induced high neutralizing antibody titers against the WT isolate of the virus, and, with partners in India and Indonesia, it was later developed into two closely resembling human vaccines, Corbevax and Indovac. Here, we describe the development and characterization of a next-generation vaccine adapted to the recently emerging XBB variants of SARS-CoV-2. (2) Methods: We conducted preclinical studies in mice using a novel yeast-produced SARS-CoV-2 XBB.1.5 RBD subunit vaccine candidate formulated with alum and CpG. We examined the neutralization profile of sera obtained from mice vaccinated twice intramuscularly at a 21-day interval with the XBB.1.5-based RBD vaccine, against WT, Beta, Delta, BA.4, BQ.1.1, BA.2.75.2, XBB.1.16, XBB.1.5, and EG.5.1 SARS-CoV-2 pseudoviruses. (3) Results: The XBB.1.5 RBD/CpG/alum vaccine elicited a robust antibody response in mice. Furthermore, the serum from vaccinated mice demonstrated potent neutralization against the XBB.1.5 pseudovirus as well as several other Omicron pseudoviruses. However, regardless of the high antibody cross-reactivity with ELISA, the anti-XBB.1.5 RBD antigen serum showed low neutralizing titers against the WT and Delta virus variants. (4) Conclusions: Whereas we observed modest cross-neutralization against Omicron subvariants with the sera from mice vaccinated with the WT RBD/CpG/Alum vaccine or with the BA.4/5-based vaccine, the sera raised against the XBB.1.5 RBD showed robust cross-neutralization. These findings underscore the imminent opportunity for an updated vaccine formulation utilizing the XBB.1.5 RBD antigen.

There is an urgent need for an accessible and low-cost COVID-19 vaccine suitable for low- and middle-income countries. Here, we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed at high levels in yeast (Pichia pastoris), as a suitable vaccine candidate against COVID-19. After introducing two modifications into the wild-type RBD gene to reduce yeast-derived hyperglycosylation and improve stability during protein expression, we show that the recombinant protein, RBD219-N1C1, is equivalent to the wild-type RBD recombinant protein (RBD219-WT) in an in vitro ACE-2 binding assay. Immunogenicity studies of RBD219-N1C1 and RBD219-WT proteins formulated with Alhydrogel® were conducted in mice, and, after two doses, both the RBD219-WT and RBD219-N1C1 vaccines induced high levels of binding IgG antibodies. Using a SARS-CoV-2 pseudovirus, we further showed that sera obtained after a two-dose immunization schedule of the vaccines were sufficient to elicit strong neutralizing antibody titers in the 1:1,000 to 1:10,000 range, for both antigens tested. The vaccines induced IFN-γ IL-6, and IL-10 secretion, among other cytokines. Overall, these data suggest that the RBD219-N1C1 recombinant protein, produced in yeast, is suitable for further evaluation as a human COVID-19 vaccine, in particular, in an Alhydrogel® containing formulation and possibly in combination with other immunostimulants.

Tc24-C4, a modified recombinant flagellar calcium-binding protein of Trypanosoma cruzi, is under development as a therapeutic subunit vaccine candidate to prevent or delay progression of chronic Chagasic cardiomyopathy. When combined with Toll-like receptor agonists, Tc24-C4 immunization reduces parasitemia, parasites in cardiac tissue, and cardiac fibrosis and inflammation in animal models. To support further research on the vaccine candidate and its mechanism of action, murine monoclonal antibodies (mAbs) against Tc24-C4 were generated. Here, we report new findings made with mAb Tc24-C4/884 that detects Tc24-WT and Tc24-C4, as well as native Tc24 in T. cruzi on ELISA, western blots, and different imaging techniques. Surprisingly, detection of Tc24 by Tc24-C/884 in fixed T. cruzi trypomastigotes required permeabilization of the parasite, revealing that Tc24 is not exposed on the surface of T. cruzi, making a direct role of antibodies in the induced protection after Tc24-C4 immunization less likely. We further observed that after immunostaining T. cruzi-infected cells with mAb Tc24-C4/884, the expression of Tc24 decreases significantly when T. cruzi trypomastigotes enter host cells and transform into amastigotes. However, Tc24 is then upregulated in association with parasite flagellar growth linked to re-transformation into the trypomastigote form, prior to host cellular escape. These observations are discussed in the context of potential mechanisms of vaccine immunity.

Chronic Chagasic cardiomyopathy (CCC) is a severe clinical manifestation that develops in 30%–40% of individuals chronically infected with the protozoal parasite Trypanosoma cruzi and is thus an important public health problem. Parasite persistence during chronic infection drives pathologic changes in the heart, including myocardial inflammation and progressive fibrosis, that contribute to clinical disease. Clinical manifestations of CCC span a range of symptoms, including cardiac arrhythmias, thromboembolic disease, dilated cardiomyopathy, and heart failure. This study aimed to investigate the role of signal transducer and activator of transcription-3 (STAT3) in cardiac pathology in a mouse model of CCC. STAT3 is a known cellular mediator of collagen deposition and fibrosis. Mice were infected with T. cruzi and then treated daily from 70 to 91 days post infection (DPI) with TTI-101, a small molecule inhibitor of STAT3; benznidazole; a combination of benznidazole and TTI-101; or vehicle alone. Cardiac function was evaluated at the beginning and end of treatment by echocardiography. By the end of treatment, STAT3 inhibition with TTI-101 eliminated cardiac fibrosis and fibrosis biomarkers but increased cardiac inflammation; serum levels of interleukin-6 (IL-6), and IFN−γ; cardiac gene expression of STAT1 and nuclear factor-κB (NF-κB); and upregulation of IL-6 and Type I and Type II IFN responses. Concurrently, decreased heart function was measured by echocardiography and myocardial strain. These results indicate that STAT3 plays a critical role in the cardiac inflammatory–fibrotic axis during CCC.

The eradication of the vector Rhodnius prolixus from Central America was heralded as a victory for controlling transmission of Trypanosoma cruzi, the parasite that causes Chagas disease. While public health officials believed this milestone achievement would effectively eliminate Chagas disease, case reports of acute vector transmission began amassing within a few years. This investigation employed a cross-sectional serosurvey of children either presenting with fever for clinical care or children living in homes with known triatomine presence in the state of Sonsonate, El Salvador. Over the 2018 calendar year, a 2.3% Chagas disease seroprevalence among children with hotspot clustering in Nahuizalco was identified. Positive serology was significantly associated with dogs in the home, older participant age, and a higher number of children in the home by multivariate regression. Concomitant intestinal parasitic infection was noted in a subset of studied children; 60% having at least one intestinal parasite and 15% having two or more concomitant infections. Concomitant parasitic infection was statistically associated with an overall higher parasitic load detected in stool by qPCR. Lastly, a four-fold higher burden of stunting was identified in the cohort compared to the national average, with four-fifths of mothers reporting severe food insecurity. This study highlights that polyparasitism is common, and a systems-based approach is warranted when treating Chagas disease seropositive children.

Background: Dengue is an infectious disease, is transmitted by the Aedes aegypti mosquito, especially in tropical regions. In the last few years, the city of Posadas (Misiones-Argentina) faces an increase in the number of cases that has led the provincial government to decree a sanitary and epidemiological emergency in 2020. Objective: To analyze the dengue social representations, present in the two newspaper publications of Misiones-Argentina relating the results to a predecessor study. Methodology: Cross-sectional observational study with a descriptive design, in which the Atlas.Ti program was used to analyze 46 news articles published by two local newspapers (El Territorio and Primera Edición) on "dengue" where the biggest outbreak occurs between April and July 2023. Results: In the publications it exists a predominance of the biomedical perspective of the disease, information evidences epidemiological data, symptomatology of the disease, and, in a lesser manner, health education or ecoepidemiology, which are necessary for health policies, were dealt with. Conclusions: The population needs tools for prevention and newspaper articles disseminate domestic and community measures to deal with the problematic; sociocommunity information is relegated by the biomedical perspective that influences in social representations.

Abstract Chagas disease resulting from Trypanosoma cruzi infection leads to a silent, long-lasting chronic neglected tropical disease affecting the poorest and underserved populations around the world. Antiparasitic treatment with benznidazole does not prevent disease progression or death in patients with established cardiac disease. Our consortium is developing a therapeutic vaccine based on the T. cruzi flagellar—derived antigen Tc24-C4 formulated with a Toll-like receptor 4 agonist adjuvant, to complement existing chemotherapy and improve treatment efficacy. Here we demonstrate that therapeutic treatment of acutely infected mice with a reduced dose of benznidazole concurrently with vaccine treatment – also known as “vaccine-linked chemotherapy”—induced a TH17 like immune response, with significantly increased production of antigen specific IL-17A, IL-23 and IL-22, and CD8 + T lymphocytes, as well as significantly increased T. cruzi specific IFNγ-producing CD4 + T lymphocytes. Significantly reduced cardiac inflammation, fibrosis, and parasite burdens and improved survival were achieved by vaccine-linked chemotherapy and individual treatments. Importantly, low dose treatments were comparably efficacious to high dose treatments, demonstrating potential dose sparing effects. We conclude that through induction of TH17 immune responses vaccine-linked chemotherapeutic strategies could bridge the tolerability and efficacy gaps of current drug treatment in Chagasic patients.

PurposesMost molecular-based published studies on breast cancer do not adequately represent the unique and diverse genetic admixture of the Latin American population. Searching for similarities and differences in molecular pathways associated with these tumors and evaluating its impact on prognosis may help to select better therapeutic approaches.Patients and MethodsWe collected clinical, pathological, and transcriptomic data of a multi-country Latin American cohort of 1,071 stage II-III breast cancer patients of the Molecular Profile of Breast Cancer Study (MPBCS) cohort. The 5-year prognostic ability of intrinsic (transcriptomic-based) PAM50 and immunohistochemical classifications, both at the cancer-specific (OSC) and disease-free survival (DFS) stages, was compared. Pathway analyses (GSEA, GSVA and MetaCore) were performed to explore differences among intrinsic subtypes.ResultsPAM50 classification of the MPBCS cohort defined 42·6% of tumors as LumA, 21·3% as LumB, 13·3% as HER2E and 16·6% as Basal. Both OSC and DFS for LumA tumors were significantly better than for other subtypes, while Basal tumors had the worst prognosis. While the prognostic power of traditional subtypes calculated with hormone receptors (HR), HER2 and Ki67 determinations showed an acceptable performance, PAM50-derived risk of recurrence best discriminated low, intermediate and high-risk groups. Transcriptomic pathway analysis showed high proliferation (i.e. cell cycle control and DNA damage repair) associated with LumB, HER2E and Basal tumors, and a strong dependency on the estrogen pathway for LumA. Terms related to both innate and adaptive immune responses were seen predominantly upregulated in Basal tumors, and, to a lesser extent, in HER2E, with respect to LumA and B tumors.ConclusionsThis is the first study that assesses molecular features at the transcriptomic level in a multicountry Latin American breast cancer patient cohort. Hormone-related and proliferation pathways that predominate in PAM50 and other breast cancer molecular classifications are also the main tumor-driving mechanisms in this cohort and have prognostic power. The immune-related features seen in the most aggressive subtypes may pave the way for therapeutic approaches not yet disseminated in Latin America.Clinical Trial RegistrationClinicalTrials.gov (Identifier: NCT02326857).

Abstract Background According to the projections of the World Health Organization, 15% of all disabilities will be associated with mental illnesses by 2020. One of the mental disorders with the largest social impacts due to high personal and family costs is psychosis. Among the most effective psychological approaches to treat schizophrenia and other psychotic disorders at the world level is cognitive behavioral therapy. Recently, cognitive behavioral therapy has introduced several tools and strategies that promote psychological processes based on acceptance and mindfulness. A large number of studies support the effectiveness of mindfulness in dealing with various mental health problems, including psychosis. This study is aimed at determining the efficiency of a mindfulness-based program in increasing cognitive function and psychological well-being in patients with a first episode of schizophrenia and a high risk mental state (those at risk of developing an episode of psychosis). Methods and design This is an experimentally designed, multi-center randomized controlled trial, with a 3-month follow-up period. The study participants will be 48 patients diagnosed with schizophrenia (first episode) and 48 with a high-risk mental state, from Santiago, Chile, aged between 15 and 35 years. Participants will be submitted to a mindfulness-based intervention (MBI), which will involve taking part in eight mindfulness workshops adapted for people with psychosis. Workshops will last approximately 1.5 hours and take place once a week, over 8 weeks. The primary outcome will be the cognitive function through Measurement and Treatment Research to Improve Cognition in Schizophrenia (MATRICS) and the secondary outcome will be psychological well-being measured by self-reporting questionnaires. Discussion The outcomes of this trial will add empirical evidence to the benefits and feasibility of MBIs for the psychotherapeutic treatment of patients with schizophrenia and high-risk mental states in reducing cognitive impairment in attention, working memory, and social cognition, as well as increasing the psychological well-being by empowering the patients’ personal resources in the management of their own symptoms and psychotic experiences. Trial registration ISRCTN registration number ISRCTN24327446 . Registered on 12 September 2016.

O presente artigo aborda a relação entre a experiência religiosa e o enfrentamento de situações de aflição e adoecimento. Por meio da análise de trajetórias de conversão e adesão religiosa de famílias negras que buscavam alívio para os seus sofrimentos ou problemas de saúde, buscou-se compreender de que forma a experiência religiosa interfere no modo de conduzir a vida e no enfrentamento de situações adversas. O trabalho integra uma pesquisa mais ampla de caráter etnográfico, realizada em um bairro popular de Salvador, Bahia. A partir de entrevistas semiestruturadas e imersão em campo, constatou-se que as famílias investigadas recorriam às mais variadas religiões buscando amenizar o sofrimento físico ou espiritual, individual ou familiar, produzindo trajetórias singulares que refletem a diversidade de crenças e alternativas de cura oferecidas por instituições religiosas na contemporaneidade.