Evaluation of a rapid diagnostic test for measles IgM detection; accuracy and the reliability of visual reading using sera from the measles surveillance programme in Brazil, 2015.

Laboratory-based case confirmation is an integral part of measles surveillance programmes; however, logistical constraints can delay response. Use of RDTs during initial patient contact could enhance surveillance by real-time case confirmation and accelerating public health response. Here, we evaluate performance of a novel measles IgM RDT and assess accuracy of visual interpretation using a representative collection of 125 sera from the Brazilian measles surveillance programme. RDT results were interpreted visually by a panel of six independent observers, the consensus of three observers and by relative reflectance measurements using an ESEQuant Reader. Compared to the Siemens anti-measles IgM EIA, sensitivity and specificity of the RDT were 94.9% (74/78, 87.4-98.6%) and 95.7% (45/47, 85.5-99.5%) for consensus visual results, and 93.6% (73/78, 85.7-97.9%) and 95.7% (45/47, 85.5-99.5%), for ESEQuant measurement, respectively. Observer agreement, determined by comparison between individuals and visual consensus results, and between individuals and ESEQuant measurements, achieved average kappa scores of 0.97 and 0.93 respectively. The RDT has the sensitivity and specificity required of a field-based test for measles diagnosis, and high kappa scores indicate this can be accomplished accurately by visual interpretation alone. Detailed studies are needed to establish its role within the global measles control programme.

Morphology and morphogenesis of SARS-CoV-2 in Vero-E6 cells.

BACKGROUND: The coronaviruses (CoVs) called the attention of the world for causing outbreaks of severe acute respiratory syndrome (SARS-CoV), in Asia in 2002-03, and respiratory disease in the Middle East (MERS-CoV), in 2012. In December 2019, yet again a new coronavirus (SARS-CoV-2) first identified in Wuhan, China, was associated with a severe respiratory infection, known today as COVID-19. This new virus quickly spread throughout China and 30 additional countries. As result, the World Health Organization (WHO) elevated the status of the COVID-19 outbreak from emergency of international concern to pandemic on March 11, 2020. The impact of COVID-19 on public health and economy fueled a worldwide race to approve therapeutic and prophylactic agents, but so far, there are no specific antiviral drugs or vaccines available. In current scenario, the development of in vitro systems for viral mass production and for testing antiviral and vaccine candidates proves to be an urgent matter. OBJECTIVE: The objective of this paper is study the biology of SARS-CoV-2 in Vero-E6 cells at the ultrastructural level. METHODS: In this study, we documented, by transmission electron microscopy and real-time reverse transcription polymerase chain reaction (RT-PCR), the infection of Vero-E6 cells with SARS-CoV-2 samples isolated from Brazilian patients. FINDINGS: The infected cells presented cytopathic effects and SARS-CoV-2 particles were observed attached to the cell surface and inside cytoplasmic vesicles. The entry of the virus into cells occurred through the endocytic pathway or by fusion of the viral envelope with the cell membrane. Assembled nucleocapsids were verified inside rough endoplasmic reticulum cisterns (RER). Viral maturation seemed to occur by budding of viral particles from the RER into smooth membrane vesicles. MAIN CONCLUSIONS: Therefore, the susceptibility of Vero-E6 cells to SARS-CoV-2 infection and the viral pathway inside the cells were demonstrated by ultrastructural analysis.

Identification of SARS-CoV-2 and additional respiratory pathogens cases under the investigation of COVID-19 initial phase in a Brazilian reference laboratory.

Coronavirus disease 2019 (COVID-19) surveillance, in Brazil, initiated shortly after its description, in China. Our aim was to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and additional pathogens in samples from the initial phase of the outbreak in Brazil, from late February to late March. From 707 samples analysed, 29 (4.1%) were SARS-CoV-2 positive. Fever and cough were their most prevalent symptoms. Co-detection of rhinovirus was observed in 2 (6.9%) cases. Additional pathogens were identified in 66.1% of the SARS-CoV-2 negative cases, mainly rhinovirus and influenza A(H1N1)pdm09. Thus, we emphasise the importance of differential diagnosis in COVID-19 suspected cases.

The Combined Deficiency of Immunoproteasome Subunits Affects Both the Magnitude and Quality of Pathogen- and Genetic Vaccination-Induced CD8+ T Cell Responses to the Human Protozoan Parasite Trypanosoma cruzi.

The ß1i, ß2i and ß5i immunoproteasome subunits have an important role in defining the repertoire of MHC class I-restricted epitopes. However, the impact of combined deficiency of the three immunoproteasome subunits in the development of protective immunity to intracellular pathogens has not been investigated. Here, we demonstrate that immunoproteasomes play a key role in host resistance and genetic vaccination-induced protection against the human pathogen Trypanosoma cruzi (the causative agent of Chagas disease), immunity to which is dependent on CD8+ T cells and IFN-γ (the classical immunoproteasome inducer). We observed that infection with T. cruzi triggers the transcription of immunoproteasome genes, both in mice and humans. Importantly, genetically vaccinated or T. cruzi-infected ß1i, ß2i and ß5i triple knockout (TKO) mice presented significantly lower frequencies and numbers of splenic CD8+ effector T cells (CD8+CD44highCD62Llow) specific for the previously characterized immunodominant (VNHRFTLV) H-2Kb-restricted T. cruzi epitope. Not only the quantity, but also the quality of parasite-specific CD8+ T cell responses was altered in TKO mice. Hence, the frequency of double-positive (IFN-γ+/TNF+) or single-positive (IFN-γ+) cells specific for the H-2Kb-restricted immunodominant as well as subdominant T. cruzi epitopes were higher in WT mice, whereas TNF single-positive cells prevailed among CD8+ T cells from TKO mice. Contrasting with their WT counterparts, TKO animals were also lethally susceptible to T. cruzi challenge, even after an otherwise protective vaccination with DNA and adenoviral vectors. We conclude that the immunoproteasome subunits are key determinants in host resistance to T. cruzi infection by influencing both the magnitude and quality of CD8+ T cell responses.

Whole-Genome Characterization of a Novel Human Influenza A(H1N2) Virus Variant, Brazil.

We report the characterization of a novel reassortant influenza A(H1N2) virus not previously reported in humans. Recovered from a a pig farm worker in southeast Brazil who had influenza-like illness, this virus is a triple reassortant containing gene segments from subtypes H1N2 (hemagglutinin), H3N2 (neuraminidase), and pandemic H1N1 (remaining genes).

A human type 5 adenovirus-based Trypanosoma cruzi therapeutic vaccine re-programs immune response and reverses chronic cardiomyopathy.

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is a prototypical neglected tropical disease. Specific immunity promotes acute phase survival. Nevertheless, one-third of CD patients develop chronic chagasic cardiomyopathy (CCC) associated with parasite persistence and immunological unbalance. Currently, the therapeutic management of patients only mitigates CCC symptoms. Therefore, a vaccine arises as an alternative to stimulate protective immunity and thereby prevent, delay progression and even reverse CCC. We examined this hypothesis by vaccinating mice with replication-defective human Type 5 recombinant adenoviruses (rAd) carrying sequences of amastigote surface protein-2 (rAdASP2) and trans-sialidase (rAdTS) T. cruzi antigens. For prophylactic vaccination, naïve C57BL/6 mice were immunized with rAdASP2+rAdTS (rAdVax) using a homologous prime/boost protocol before challenge with the Colombian strain. For therapeutic vaccination, rAdVax administration was initiated at 120 days post-infection (dpi), when mice were afflicted by CCC. Mice were analyzed for electrical abnormalities, immune response and cardiac parasitism and tissue damage. Prophylactic immunization with rAdVax induced antibodies and H-2Kb-restricted cytotoxic and interferon (IFN)γ-producing CD8+ T-cells, reduced acute heart parasitism and electrical abnormalities in the chronic phase. Therapeutic vaccination increased survival and reduced electrical abnormalities after the prime (analysis at 160 dpi) and the boost (analysis at 180 and 230 dpi). Post-therapy mice exhibited less heart injury and electrical abnormalities compared with pre-therapy mice. rAdVax therapeutic vaccination preserved specific IFNγ-mediated immunity but reduced the response to polyclonal stimuli (anti-CD3 plus anti-CD28), CD107a+ CD8+ T-cell frequency and plasma nitric oxide (NO) levels. Moreover, therapeutic rAdVax reshaped immunity in the heart tissue as reduced the number of perforin+ cells, preserved the number of IFNγ+ cells, increased the expression of IFNγ mRNA but reduced inducible NO synthase mRNA. Vaccine-based immunostimulation with rAd might offer a rational alternative for re-programming the immune response to preserve and, moreover, recover tissue injury in Chagas' heart disease.

Apoptosis-associated speck-like protein containing a caspase recruitment domain inflammasomes mediate IL-1ß response and host resistance to Trypanosoma cruzi infection.

The innate immune response to Trypanosoma cruzi infection comprises several pattern recognition receptors (PRRs), including TLR-2, -4, -7, and -9, as well as the cytosolic receptor Nod1. However, there are additional PRRs that account for the host immune responses to T. cruzi. In this context, the nucleotide-binding oligomerization domain-like receptors (NLRs) that activate the inflammasomes are candidate receptors that deserve renewed investigation. Following pathogen infection, NLRs form large molecular platforms, termed inflammasomes, which activate caspase-1 and induce the production of active IL-1ß and IL-18. In this study, we evaluated the involvement of inflammasomes in T. cruzi infection and demonstrated that apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) inflammasomes, including NLR family, pyrin domain-containing 3 (NLRP3), but not NLR family, caspase recruitment domain-containing 4 or NLR family, pyrin domain-containing 6, are required for triggering the activation of caspase-1 and the secretion of IL-1ß. The mechanism by which T. cruzi mediates the activation of the ASC/NLRP3 pathway involves K⁺ efflux, lysosomal acidification, reactive oxygen species generation, and lysosomal damage. We also demonstrate that despite normal IFN-γ production in the heart, ASC⁻/⁻ and caspase-1⁻/⁻ infected mice exhibit a higher incidence of mortality, cardiac parasitism, and heart inflammation. These data suggest that ASC inflammasomes are critical determinants of host resistance to infection with T. cruzi.

UNC93B1 and nucleic acid-sensing Toll-like receptors mediate host resistance to infection with Leishmania major.

The mammalian homolog B1 of Unc-93 Caenorhabditis elegans known as UNC93B1 is a chaperone protein that mediates translocation of the nucleic acid-sensing Toll-like receptors (TLRs) from the endoplasmic reticulum to the endolysosomes. The triple deficient (UNC93B1 mutant) mice have a functional single point mutation in the UNC93B1 that results in non-functional TLR3, TLR7, and TLR9. Herein, we demonstrate that UNC93B1 mutant mice, in the C57BL/6 (resistant) genetic background, are highly susceptible to Leishmania major infection. Enhanced swelling of the footpad was associated with high levels of interleukin 10, decreased levels of interferon γ, and increased parasitism. None of the single TLR3, TLR7, and TLR9 knock-out (KO) mice resemble the UNC93B1 mutant phenotype upon infection with L. major. Whereas the double TLR7/TLR9 KO showed a partial phenotype, the triple TLR3/TLR7/TLR9 KO mice were as susceptible as the UNC93B1 mutant mice, when infected with Leishmania parasites. Finally, we demonstrate that treatment with either anti-interleukin 10 receptor monoclonal antibody or recombinant interleukin 12 restored a robust anti-parasite TH1 response and reverted the susceptible phenotype of UNC93B1 mutant mice. Altogether, our results indicate the redundant and essential role of nucleic acid-sensing TLR3, TLR7 and TLR9 in inducing interleukin 12, development of a TH1 response, and resistance to L. major infection in resistant C57BL/6 mice.

Requirement of UNC93B1 reveals a critical role for TLR7 in host resistance to primary infection with Trypanosoma cruzi.

UNC93B1 associates with TLR3, 7, and 9, mediating their translocation from the endoplasmic reticulum to the endolysosome, thus allowing proper activation by microbial nucleic acids. We found that the triple-deficient 3d mice, which lack functional UNC93B1 as well as functional endosomal TLRs, are highly susceptible to infection with Trypanosoma cruzi. The enhanced parasitemia and mortality in 3d animals were associated with impaired proinflammatory response, including reduced levels of IL-12p40 and IFN-γ. Importantly, the phenotype of 3d mice was intermediary between MyD88(-/-) (highly susceptible) and TLR9(-/-) (moderately susceptible), indicating the involvement of an additional UN93B1-dependent TLR(s) on host resistance to T. cruzi. Hence, our experiments also revealed that TLR7 is a critical innate immune receptor involved in recognition of parasite RNA, induction of IL-12p40 by dendritic cells, and consequent IFN-γ by T lymphocytes. Furthermore, we show that upon T. cruzi infection, triple TLR3/7/9(-/-) mice had similar phenotype than 3d mice. These data imply that the nucleic acid-sensing TLRs are critical determinants of host resistance to primary infection with T. cruzi.

Human Brain Microvascular Endothelial Cells Exposure to SARS-CoV-2 Leads to Inflammatory Activation through NF-κB Non-Canonical Pathway and Mitochondrial Remodeling.

Neurological effects of COVID-19 and long-COVID-19, as well as neuroinvasion by SARS-CoV-2, still pose several questions and are of both clinical and scientific relevance. We described the cellular and molecular effects of the human brain microvascular endothelial cells (HBMECs) in vitro exposure by SARS-CoV-2 to understand the underlying mechanisms of viral transmigration through the blood-brain barrier. Despite the low to non-productive viral replication, SARS-CoV-2-exposed cultures displayed increased immunoreactivity for cleaved caspase-3, an indicator of apoptotic cell death, tight junction protein expression, and immunolocalization. Transcriptomic profiling of SARS-CoV-2-challenged cultures revealed endothelial activation via NF-κB non-canonical pathway, including RELB overexpression and mitochondrial dysfunction. Additionally, SARS-CoV-2 led to altered secretion of key angiogenic factors and to significant changes in mitochondrial dynamics, with increased mitofusin-2 expression and increased mitochondrial networks. Endothelial activation and remodeling can further contribute to neuroinflammatory processes and lead to further BBB permeability in COVID-19.

Results from the second WHO external quality assessment for the molecular detection of respiratory syncytial virus, 2019-2020.

Background: External quality assessments (EQAs) for the molecular detection of human respiratory syncytial virus (RSV) are necessary to ensure the standardisation of reliable results. The Phase II, 2019-2020 World Health Organization (WHO) RSV EQA included 28 laboratories in 26 countries. The EQA panel evaluated performance in the molecular detection and subtyping of RSV-A and RSV-B. This manuscript describes the preparation, distribution, and analysis of the 2019-2020 WHO RSV EQA. Methods: Panel isolates underwent whole genome sequencing and in silico primer matching. The final panel included nine contemporary, one historical virus and two negative controls. The EQA panel was manufactured and distributed by the UK National External Quality Assessment Service (UK NEQAS). National laboratories used WHO reference assays developed by the United States Centers for Disease Control and Prevention, an RSV subtyping assay developed by the Victorian Infectious Diseases Reference Laboratory (Australia), or other in-house or commercial assays already in use at their laboratories. Results: An in silico analysis of isolates showed a good match to assay primer/probes. The panel was distributed to 28 laboratories. Isolates were correctly identified in 98% of samples for detection and 99.6% for subtyping. Conclusions: The WHO RSV EQA 2019-2020 showed that laboratories performed at high standards. Updating the composition of RSV molecular EQAs with contemporary strains to ensure representation of circulating strains, and ensuring primer matching with EQA panel viruses, is advantageous in assessing diagnostic competencies of laboratories. Ongoing EQAs are recommended because of continued evolution of mismatches between current circulating strains and existing primer sets.

Intrinsic expression of Nod2 in CD4+ T lymphocytes is not necessary for the development of cell-mediated immunity and host resistance to Toxoplasma gondii.

Nod2 belongs to the nucleotide-binding domain leucine-rich repeat family of proteins and senses bacterial cell wall components to initiate innate immune responses against various pathogens. Recently, it has been reported that T-cell-intrinsic expression of Nod2 promotes host defense against Toxoplasma gondii infection by inducing type 1 immunity. Here, we present results that demonstrate that Nod2 does not play a role in the defense against T. gondii infection. Nod2-deficient mice were fully capable of inducing Th1 immune responses and did not show enhanced susceptibility to infection. Upon TCR stimulation in vitro, Nod2-deficient CD4(+) T cells showed normal activation, IL-2 production, proliferation, and Th1/2 differentiation. Nod2 mRNA and protein were expressed in CD4(+) T and CD8(+) T cells at substantial levels. Therefore, Nod2, although expressed in CD4(+) T cells, does not have an intrinsic function in T-cell activation and differentiation.

Analysis of Viral and Host Factors on Immunogenicity of 2018, 2019, and 2020 Southern Hemisphere Seasonal Trivalent Inactivated Influenza Vaccine in Adults in Brazil.

Annual vaccination against influenza is the best tool to prevent deaths and hospitalizations. Regular updates of trivalent inactivated influenza vaccines (TIV) are necessary due to high mutation rates in influenza viruses. TIV effectiveness is affected by antigenic mismatches, age, previous immunity, and other host factors. Studying TIV effectiveness annually in different populations is critical. The serological responses to Southern-Hemisphere TIV and circulating influenza strains were evaluated in 2018−2020 among Brazilian volunteers, using hemagglutination inhibition (HI) assays. Post-vaccination titers were corrected to account for pre-vaccination titers. Our population achieved >83% post-vaccination seroprotection levels, whereas seroconversion rates ranged from 10% to 46%. TIV significantly enhanced antibody titers and seroprotection against all prior and contemporary vaccine and circulating strains tested. Strong cross-reactive responses were detected, especially between H1N1 subtypes. A/Singapore/INFIMH-16-0019/2016, included in the 2018 TIV, induced the poorest response. Significant titer and seroprotection reductions were observed 6 and 12 months after vaccination. Age had a slight effect on TIV response, whereas previous vaccination was associated with lower seroconversion rates and titers. Despite this, TIV induced high seroprotection for all strains, in all groups. Regular TIV evaluations, based on regional influenza strain circulation, should be conducted and the factors affecting response studied.

Sera of patients infected by earlier lineages of SARS-CoV-2 are capable to neutralize later emerged variants of concern.

Serum samples of 20 hospitalized coronavirus disease 2019 (COVID-19) patients from Brazil who were infected by the earlier severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages B.1.1.28 and B.1.1.33, and by the variant of concern (VOC) Gamma (P.1) were tested by plaque reduction neutralization test (PRNT90) with wild isolates of a panel of SARS-CoV-2 lineages, including B.1, Zeta, N.10, and the VOCs Gamma, Alpha, and Delta that emerged in different timeframes of the pandemic. The main objective of this study was to evaluate if the serum of patients infected by earlier lineages was capable to neutralize later emerged VOCs. We also evaluated if the 4-fold difference in PRNT90 titers is a reliable seropositivity criterion to distinguish infections caused by different SARS-CoV-2 lineages. Sera collected between May 2020 and August 2021 from the day of admittance to the hospital to 21 days after diagnostic of patients infected by the two earlier lineages B.1.1.28 and B.1.1.33 presented neutralizing capacity for all challenged VOCs, including Gamma and Delta. Among all variants tested, Delta and N.10 presented the lowest geometric mean of neutralizing antibody titers, and B.1.1.7, presented the highest titers. Four patients infected with Gamma, that emerged in December 2020, presented neutralizing antibodies for B.1, B.1.1.33, and B.1.1.28, its ancestor lineage. All of them had neutralizing antibodies under the level of detection for the VOC Delta. Patients infected by B.1.1.28 presented very similar geometric mean of neutralizing antibody titers for both B.1.1.33 and B.1.1.28. Findings presented here indicate that most patients infected in early stages of COVID-19 pandemic presented neutralizing antibodies capable to neutralize wild types of all later emerged VOCs in Brazil, and that the 4-fold difference in PRNT90 titers is not reliable to distinguish humoral response among different SARS-CoV-2 lineages.

SARS-CoV-2 infection of human brain microvascular endothelial cells leads to inflammatory activation through NF-κB non-canonical pathway and mitochondrial remodeling.

Neurological effects of COVID-19 and long-COVID-19 as well as neuroinvasion by SARS-CoV-2 still pose several questions and are of both clinical and scientific relevance. We described the cellular and molecular effects of the human brain microvascular endothelial cells (HBMECs) in vitro infection by SARS-CoV-2 to understand the underlying mechanisms of viral transmigration through the Blood-Brain Barrier. Despite the low to non-productive viral replication, SARS-CoV-2-infected cultures displayed increased apoptotic cell death and tight junction protein expression and immunolocalization. Transcriptomic profiling of infected cultures revealed endothelial activation via NF-κB non-canonical pathway, including RELB overexpression, and mitochondrial dysfunction. Additionally, SARS-CoV-2 led to altered secretion of key angiogenic factors and to significant changes in mitochondrial dynamics, with increased mitofusin-2 expression and increased mitochondrial networks. Endothelial activation and remodeling can further contribute to neuroinflammatory processes and lead to further BBB permeability in COVID-19.

SARS-CoV-2 infection of human brain microvascular endothelial cells leads to inflammatory activation through NF-κB non-canonical pathway and mitochondrial remodeling.

Neurological effects of COVID-19 and long-COVID-19 as well as neuroinvasion by SARS-CoV-2 still pose several questions and are of both clinical and scientific relevance. We described the cellular and molecular effects of the human brain microvascular endothelial cells (HBMECs) in vitro infection by SARS-CoV-2 to understand the underlying mechanisms of viral transmigration through the Blood-Brain Barrier. Despite the low to non- productive viral replication, SARS-CoV-2-infected cultures displayed increased apoptotic cell death and tight junction protein expression and immunolocalization. Transcriptomic profiling of infected cultures revealed endothelial activation via NF-κB non-canonical pathway, including RELB overexpression, and mitochondrial dysfunction. Additionally, SARS-CoV-2 led to altered secretion of key angiogenic factors and to significant changes in mitochondrial dynamics, with increased mitofusin-2 expression and increased mitochondrial networks. Endothelial activation and remodeling can further contribute to neuroinflammatory processes and lead to further BBB permeability in COVID-19.

Identification of Hypericin as a Candidate Repurposed Therapeutic Agent for COVID-19 and Its Potential Anti-SARS-CoV-2 Activity.

The COVID-19 pandemic has had an unprecedented impact on the global economy and public health. Its etiologic agent, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible, pathogenic and has a rapid global spread. Currently, the increase in the number of new confirmed cases has been slowed down due to the increase of vaccination in some regions of the world. Still, the rise of new variants has influenced the detection of additional waves of rising cases that some countries have experienced. Since the virus replication cycle is composed of many distinct stages, some viral proteins related to them, as the main-protease (Mpro) and RNA dependent RNA polymerase (RdRp), constitute individual potential antiviral targets. In this study, we challenged the mentioned enzymes against compounds pre-approved by health regulatory agencies in a virtual screening and later in Molecular Mechanics/Poisson-Bolzmann Surface Area (MM/PBSA) analysis. Our results showed that, among the identified potential drugs with anti-SARS-CoV-2 properties, Hypericin, an important component of the Hypericum perforatum that presents antiviral and antitumoral properties, binds with high affinity to viral Mpro and RdRp. Furthermore, we evaluated the activity of Hypericin anti-SARS-CoV-2 replication in an in vitro model of Vero-E6 infected cells. Therefore, we show that Hypericin inhibited viral replication in a dose dependent manner. Moreover, the cytotoxicity of the compound, in cultured cells, was evaluated, but no significant activity was found. Thus, the results observed in this study indicate that Hypericin is an excellent candidate for repurposing for the treatment of COVID-19, with possible inhibition of two important phases of virus maturation.

Susceptibility to re-infection in C57BL/6 mice with recombinant strains of Toxoplasma gondii.

This work reports results of re-infection of BALB/c and C57BL/6 mice with different recombinant strains of Toxoplasma gondii. Mice were prime-infected with the non-virulent D8 strain and challenged with virulent strains. PCR-RFLP of cS10-A6 genetic marker of T. gondii demonstrated that BALB/c mice were re-infected with the EGS strain, while C57BL/6 mice were re-infected with the EGS and CH3 strains. Levels of IFN-γ and IL-10 after D8 prime-infection were lower in C57BL/6 than in BALB/c mice. Brain inflammation after D8 prime-infection was more intense in C57BL/6 than in BALB/c mice. It was shown that re-infection depends on mice lineage and genotype of the strain used in the challenge.

Two-Step In Vitro Model to Evaluate the Cellular Immune Response to SARS-CoV-2.

The cellular immune response plays an important role in COVID-19, caused by SARS-CoV-2. This feature makes use of in vitro models' useful tools to evaluate vaccines and biopharmaceutical effects. Here, we developed a two-step model to evaluate the cellular immune response after SARS-CoV-2 infection-induced or spike protein stimulation in peripheral blood mononuclear cells (PBMC) from both unexposed and COVID-19 (primo-infected) individuals (Step1). Moreover, the supernatants of these cultures were used to evaluate its effects on lung cell lines (A549) (Step2). When PBMC from the unexposed were infected by SARS-CoV-2, cytotoxic natural killer and nonclassical monocytes expressing inflammatory cytokines genes were raised. The supernatant of these cells can induce apoptosis of A549 cells (mock vs. Step2 [mean]: 6.4% × 17.7%). Meanwhile, PBMCs from primo-infected presented their memory CD4+ T cells activated with a high production of IFNG and antiviral genes. Supernatant from past COVID-19 subjects contributed to reduce apoptosis (mock vs. Step2 [ratio]: 7.2 × 1.4) and to elevate the antiviral activity (iNOS) of A549 cells (mock vs. Step2 [mean]: 31.5% × 55.7%). Our findings showed features of immune primary cells and lung cell lines response after SARS-CoV-2 or spike protein stimulation that can be used as an in vitro model to study the immunity effects after SARS-CoV-2 antigen exposure.

The endless race between Trypanosoma cruzi and host immunity: lessons for and beyond Chagas disease.

Infection with the protozoan parasite Trypanosoma cruzi, the agent of Chagas disease, is characterised by a variable clinical course - from symptomless cases to severe chronic disease with cardiac and/or gastrointestinal involvement. The variability in disease outcome has been attributed to host responses as well as parasite heterogeneity. In this article, we review studies indicating the importance of immune responses as key determinants of host resistance to T. cruzi infection and the pathogenesis of Chagas disease. Particular attention is given to recent studies defining the role of cognate innate immune receptors and immunodominant CD8+ T cells that recognise parasite components - both crucial for host-parasite interaction and disease outcome. In light of these studies we speculate about parasite strategies that induce a strong and long-lasting T-cell-mediated immunity but at the same time allow persistence of the parasite in the vertebrate host. We also discuss what we have learned from these studies for increasing our understanding of Chagas pathogenesis and for the design of new strategies to prevent the development of Chagas disease. Finally, we highlight recent studies employing a genetically engineered attenuated T. cruzi strain as a vaccine shuttle that elicits potent T cell responses specific to a tumour antigen and protective immunity against a syngeneic melanoma cell line.