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 beta1i, beta2i and beta5i 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-gamma (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 beta1i, beta2i and beta5i 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-gamma+/TNF+) or single-positive (IFN-gamma+) 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

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

AbstractThis 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

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

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.Ludwig Institute of Cancer Research (LICR)Atlantic Philanthropies (AP)Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)Fundacao Oswaldo Cruz (FIOCRUZ)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)US National Institutes of Health (NIH)Fundacao Oswaldo Cruz, Lab Immunopatol, Ctr Pesquisas Rene Rachou, BR-30190002 Belo Horizonte, MG, BrazilUniv Fed Minas Gerais, Dept Bioquim & Imunol, Belo Horizonte, MG, BrazilUniv Massachusetts, Sch Med, Div Infect Dis & Immunol, Worcester, MA 01605 USAUniv Fed Minas Gerais, Dept Parasitol, Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Ctr Interdisciplinar Terapia Genica CINTERGEN, São Paulo, BrazilUniversidade Federal de São Paulo, Ctr Interdisciplinar Terapia Genica CINTERGEN, São Paulo, BrazilWeb of Scienc

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 gamma, 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

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

Global Influenza Hospital-based Surveillance Network (GIHSN): results of surveillance of influenza and other respiratory viruses in hospitalised patients in Brazil, 2015

Submitted by Sandra Infurna ([email protected]) on 2018-09-13T16:16:31Z No. of bitstreams: 1 marilda_siqueira_etal_IOC_2018.pdf: 797399 bytes, checksum: d8be7980d447e97f432a1230d0049417 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2018-09-13T16:34:34Z (GMT) No. of bitstreams: 1 marilda_siqueira_etal_IOC_2018.pdf: 797399 bytes, checksum: d8be7980d447e97f432a1230d0049417 (MD5)Made available in DSpace on 2018-09-13T16:34:34Z (GMT). No. of bitstreams: 1 marilda_siqueira_etal_IOC_2018.pdf: 797399 bytes, checksum: d8be7980d447e97f432a1230d0049417 (MD5) Previous issue date: 2018Universidade Federal do Paraná. Departamento de Doenças Infecciosas. Curitiba, PR, Brasil / Universidade Federal do Paraná. Laboratório de Virologia. Curitiba, PR, Brasil.Universidade Federal do Ceará. Departamento de Patologia e Medicina Legal. Fortaleza, CE, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Vírus respiratório e do Sarampo. Rio de Janeiro, RJ, Brasil.Universidade Federal do Paraná. Programa de Pós-graduação em Medicina Interna e Ciências da Saúde. Curitiba, PR, Brasil.Universidade Federal do Paraná. Laboratório de Virologia. Curitiba, PR, Brasil.Universidade Federal do Paraná. Laboratório de Virologia. Curitiba, PR, Brasil.Universidade Federal do Paraná. Laboratório de Virologia. Curitiba, PR, Brasil.Hospital Quinta D'Or. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Nacional de Infectologia. Rio de Janeiro, RJ, Brasil.Fondation Merieux. Scientific Department. Lyon, France.Fondation Merieux. Scientific Department. Lyon, France.FISABIO-Public Health. Vaccines Research. Valencia, Spain.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Vírus respiratório e do Sarampo. Rio de Janeiro, RJ, Brasil.Global Influenza Hospital-based Surveillance NetworkBackground Influenza-like illness occurs annually worldwide, with peak timing and severity varying seasonally, resulting in significant annual mortality. Objectives There were three objectives: (1) to describe the epidemiological and clinical features of hospitalised patients with severe acute respiratory infection caused by influenza and other respiratory viruses (ORVs); (2) to report the influenza seasonality in the region and (3) to correlate findings of influenza circulation and immunisation time in Brazil. Patients/methods This study took place in three Brazilian hospitals located in cities with different climatic conditions (Curitiba (south), Rio de Janeiro (south-east) and Fortaleza (north-east)). Patients presenting with an acute process with indication for admission consisting of a predefined set of conditions potentially associated with recent influenza infection were enrolled. Results We screened 1666 patients, with 595 meeting the inclusion criteria. Influenza viruses and ORVs were detected in 6.5% and 59% of patients, respectively. Influenza-positive cases fell into the severe spectrum as compared with those with ORVs (30% vs 11%), but without any difference in mortality rates. Epidemiological results revealed variations in the peak time of influenza infections between north-east (Fortaleza) and south (Curitiba) Brazil, basically following the rain period of each region. In north-east Brazil, viral circulation was prevalent in the first 4 months of the year, indicating that the vaccination campaign occurred in a postseasonal period, possibly explaining the low effectiveness. Conclusions The active-surveillance model is a valuable tool for investigating respiratory virus impact on hospitalised patients, with influenza-infection monitoring enabling implementation of adequate preventive measures

Apoptosis-associated speck-like protein containing a caspase recruitment domain inflammasomes mediate IL-1beta 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-1beta 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-1beta. 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-gamma 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