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The activation of natural killer (NK) cells depends on a change in the balance of signals from inhibitory and activating receptors. The activation threshold values of NK cells are thought to be set by engagement of inhibitory receptors during development. Here, we found that the activating receptor NKG2D specifically set the activation threshold for the activating receptor NCR1 through a process that required the adaptor DAP12. As a result, NKGD2-deficient (Klrk1-/-) mice controlled tumors and cytomegalovirus infection better than wild-type controls through the NCR1-induced production of the cytokine IFN-γ. Expression of NKG2D before the immature NK cell stage increased expression of the adaptor CD3ζ. Reduced expression of CD3ζ in Klrk1-/- mice was associated with enhanced signal transduction through NCR1, and CD3ζ deficiency resulted in hyper-responsiveness to stimulation via NCR1. Thus, an activating receptor developmentally set the activity of another activating receptor on NK cells and determined NK cell reactivity to cellular threats.
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Antígenos Ly/imunologia , Citotoxicidade Imunológica/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Receptor 1 Desencadeador da Citotoxicidade Natural/imunologia , Animais , Camundongos , Camundongos KnockoutRESUMO
Viral infections during pregnancy are a considerable cause of adverse outcomes and birth defects, and the underlying mechanisms are poorly understood. Among those, cytomegalovirus (CMV) infection stands out as the most common intrauterine infection in humans, putatively causing early pregnancy loss. We employed murine CMV as a model to study the consequences of viral infection on pregnancy outcome and fertility maintenance. Even though pregnant mice successfully controlled CMV infection, we observed highly selective, strong infection of corpus luteum (CL) cells in their ovaries. High infection densities indicated complete failure of immune control in CL cells, resulting in progesterone insufficiency and pregnancy loss. An abundance of gap junctions, absence of vasculature, strong type I interferon (IFN) responses, and interaction of innate immune cells fully protected the ovarian follicles from viral infection. Our work provides fundamental insights into the effect of CMV infection on pregnancy loss and mechanisms protecting fertility.
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Corpo Lúteo/imunologia , Infecções por Citomegalovirus/imunologia , Fertilidade/imunologia , Imunidade Inata/imunologia , Animais , Corpo Lúteo/virologia , Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Feminino , Junções Comunicantes/imunologia , Interferon Tipo I/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Gravidez , Progesterona/imunologiaRESUMO
COVID-19 vaccines prevent severe forms of the disease, but do not warrant complete protection against breakthrough infections. This could be due to suboptimal mucosal immunity at the site of virus entry, given that all currently approved vaccines are administered via the intramuscular route. In this study, we assessed humoral and cellular immune responses in BALB/c mice after intranasal and intramuscular immunization with adenoviral vector ChAdOx1-S expressing full-length Spike protein of SARS-CoV-2. We showed that both routes of vaccination induced a potent IgG antibody response, as well as robust neutralizing capacity, but intranasal vaccination elicited a superior IgA antibody titer in the sera and in the respiratory mucosa. Bronchoalveolar lavage from intranasally immunized mice efficiently neutralized SARS-CoV-2, which has not been the case in intramuscularly immunized group. Moreover, substantially higher percentages of epitope-specific CD8 T cells exhibiting a tissue resident phenotype were found in the lungs of intranasally immunized animals. Finally, both intranasal and intramuscular vaccination with ChAdOx1-S efficiently protected the mice after the challenge with recombinant herpesvirus expressing the Spike protein. Our results demonstrate that intranasal application of adenoviral vector ChAdOx1-S induces superior mucosal immunity and therefore could be a promising strategy for putting the COVID-19 pandemic under control.
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COVID-19 , Vacinas Virais , Adenoviridae/genética , Administração Intranasal , Animais , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Imunidade Celular , Imunidade nas Mucosas , Camundongos , Camundongos Endogâmicos BALB C , Pandemias/prevenção & controle , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Vacinação/métodosRESUMO
Broad tissue tropism of cytomegaloviruses (CMVs) is facilitated by different glycoprotein entry complexes, which are conserved between human CMV (HCMV) and murine CMV (MCMV). Among the wide array of cell types susceptible to the infection, mononuclear phagocytes (MNPs) play a unique role in the pathogenesis of the infection as they contribute both to the virus spread and immune control. CMVs have dedicated numerous genes for the efficient infection and evasion of macrophages and dendritic cells. In this study, we have characterized the properties and function of M116, a previously poorly described but highly transcribed MCMV gene region that encodes M116.1p, a novel protein necessary for the efficient infection of MNPs and viral spread in vivo. Our study further revealed that M116.1p shares similarities with its positional homologs in HCMV and RCMV, UL116 and R116, respectively, such as late kinetics of expression, N-glycosylation, localization to the virion assembly compartment, and interaction with gH-a member of the CMVs fusion complex. This study, therefore, expands our knowledge about virally encoded glycoproteins that play important roles in viral infectivity and tropism. IMPORTANCE Human cytomegalovirus (HCMV) is a species-specific herpesvirus that causes severe disease in immunocompromised individuals and immunologically immature neonates. Murine cytomegalovirus (MCMV) is biologically similar to HCMV, and it serves as a widely used model for studying the infection, pathogenesis, and immune responses to HCMV. In our previous work, we have identified the M116 ORF as one of the most extensively transcribed regions of the MCMV genome without an assigned function. This study shows that the M116 locus codes for a novel protein, M116.1p, which shares similarities with UL116 and R116 in HCMV and RCMV, respectively, and is required for the efficient infection of mononuclear phagocytes and virus spread in vivo. Furthermore, this study establishes the α-M116 monoclonal antibody and MCMV mutants lacking M116, generated in this work, as valuable tools for studying the role of macrophages and dendritic cells in limiting CMV infection following different MCMV administration routes.
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Sistema Fagocitário Mononuclear/virologia , Muromegalovirus/fisiologia , Proteínas do Envelope Viral/metabolismo , Animais , Fibroblastos/metabolismo , Fibroblastos/virologia , Glicosilação , Infecções por Herpesviridae/virologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Sistema Fagocitário Mononuclear/metabolismo , Transcrição Gênica , Proteínas do Envelope Viral/genética , Vírion/metabolismo , Montagem de Vírus , Internalização do Vírus , Replicação ViralRESUMO
The cloning of herpesviruses as bacterial artificial chromosomes (BACs) has revolutionized the study of herpesvirus biology, allowing rapid and precise manipulation of viral genomes. Several clinical strains of human cytomegalovirus (HCMV) have been cloned as BACs; however, no low-passage strains of murine CMV (MCMV), which provide a model mimicking these isolates, have been cloned. Here, the low-passage G4 strain of was BAC cloned. G4 carries an m157 gene that does not ligate the natural killer (NK) cell-activating receptor, Ly49H, meaning that unlike laboratory strains of MCMV, this virus replicates well in C57BL/6 mice. This BAC clone exhibited normal replication during acute infection in the spleen and liver but was attenuated for salivary gland tropism. Next-generation sequencing revealed a C-to-A mutation at nucleotide position 188422, located in the 3' untranslated region of sgg1, a spliced gene critical for salivary gland tropism. Repair of this mutation restored tropism for the salivary glands. Transcriptional analysis revealed a novel spliced gene within the sgg1 locus. This small open reading frame (ORF), sgg1.1, starts at the 3' end of the first exon of sgg1 and extends exon 2 of sgg1. This shorter spliced gene is prematurely terminated by the nonsense mutation at nt 188422. Sequence analysis of tissue culture-passaged virus demonstrated that sgg1.1 was stable, although other mutational hot spots were identified. The G4 BAC will allow in vivo studies in a broader range of mice, avoiding the strong NK cell responses seen in B6 mice with other MCMV BAC-derived MCMVs.IMPORTANCE Murine cytomegalovirus (MCMV) is widely used as a model of human CMV (HCMV) infection. However, this model relies on strains of MCMV that have been serially passaged in the laboratory for over four decades. These laboratory strains have been cloned as bacterial artificial chromosomes (BACs), which permits rapid and precise manipulation. Low-passage strains of MCMV add to the utility of the mouse model of HCMV infection but do not exist as cloned BACs. This study describes the first such low-passage MCMV BAC. This BAC-derived G4 was initially attenuated in vivo, with subsequent full genomic sequencing revealing a novel spliced transcript required for salivary gland tropism. These data suggest that MCMV, like HCMV, undergoes tissue culture adaptation that can limit in vivo growth and supports the use of BAC clones as a way of standardizing viral strains and minimizing interlaboratory strain variation.
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Cromossomos Artificiais Bacterianos/genética , Muromegalovirus/genética , Glândulas Salivares/virologia , Tropismo/fisiologia , Animais , DNA Recombinante , Feminino , Genoma Viral , Infecções por Herpesviridae/virologia , Humanos , Células Matadoras Naturais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação , Fases de Leitura Aberta , Proteínas Virais/genéticaRESUMO
Major gaps in our knowledge of pathogen genes and how these gene products interact with host gene products to cause disease represent a major obstacle to progress in vaccine and antiviral drug development for the herpesviruses. To begin to bridge these gaps, we conducted a dual analysis of Murine Cytomegalovirus (MCMV) and host cell transcriptomes during lytic infection. We analyzed the MCMV transcriptome during lytic infection using both classical cDNA cloning and sequencing of viral transcripts and next generation sequencing of transcripts (RNA-Seq). We also investigated the host transcriptome using RNA-Seq combined with differential gene expression analysis, biological pathway analysis, and gene ontology analysis. We identify numerous novel spliced and unspliced transcripts of MCMV. Unexpectedly, the most abundantly transcribed viral genes are of unknown function. We found that the most abundant viral transcript, recently identified as a noncoding RNA regulating cellular microRNAs, also codes for a novel protein. To our knowledge, this is the first viral transcript that functions both as a noncoding RNA and an mRNA. We also report that lytic infection elicits a profound cellular response in fibroblasts. Highly upregulated and induced host genes included those involved in inflammation and immunity, but also many unexpected transcription factors and host genes related to development and differentiation. Many top downregulated and repressed genes are associated with functions whose roles in infection are obscure, including host long intergenic noncoding RNAs, antisense RNAs or small nucleolar RNAs. Correspondingly, many differentially expressed genes cluster in biological pathways that may shed new light on cytomegalovirus pathogenesis. Together, these findings provide new insights into the molecular warfare at the virus-host interface and suggest new areas of research to advance the understanding and treatment of cytomegalovirus-associated diseases.
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Infecções por Herpesviridae/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , MicroRNAs/biossíntese , Muromegalovirus/fisiologia , Fatores de Transcrição/biossíntese , Transcriptoma , Regulação para Cima , Animais , Linhagem Celular Transformada , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/virologia , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/patologia , Camundongos , MicroRNAs/genética , Fatores de Transcrição/genéticaRESUMO
Cytomegalovirus (CMV), a representative member of the Betaherpesvirinae subfamily of herpesviruses, is common in the human population, but immunocompetent individuals are generally asymptomatic when infected with this virus. However, in immunocompromised individuals and immunologically immature fetuses and newborns, CMV can cause a wide range of often long-lasting morbidities and even death. CMV is not only widespread throughout the population but it is also widespread in its hosts, infecting and establishing latency in nearly all tissues and organs. Thus, understanding the pathogenesis of and immune responses to this virus is a prerequisite for developing effective prevention and treatment strategies. Multiple arms of the immune system are engaged to contain the infection, and general concepts of immune control of CMV are now reasonably well understood. Nonetheless, in recent years, tissue-specific immune responses have emerged as an essential factor for resolving CMV infection. As tissues differ in biology and function, so do immune responses to CMV and pathological processes during infection. This review discusses state-of-the-art knowledge of the immune response to CMV infection in tissues, with particular emphasis on several well-studied and most commonly affected organs.
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Congenital human cytomegalovirus (HCMV) infection may cause life-threatening disease and permanent damage to the central nervous system. The mouse model of CMV infection is most commonly used to study mechanisms of infection and pathogenesis. While essential to limit mouse CMV (MCMV) replication, the inflammatory responses, particularly IFNγ and TNFα, cause neurodevelopmental abnormalities. Other soluble mediators of the immune response in most tissues remain largely unexplored. To address this gap, we quantified 48 soluble mediators of the immune response, including 32 cytokines, 10 chemokines, 3 growth factors/regulators, and 3 soluble receptors in the spleen, liver, lungs, and brain at 9 and 14 days postinfection (dpi). Our analysis found 25 induced molecules in the brain at 9 dpi, with an additional 8 showing statistically elevated responses at 14 dpi. Specifically, all analyzed CCL group cytokines (CCL2, CCL3, CCL4, CCL5, CCL7, and CCL11) were upregulated at 14 dpi in the brain. Furthermore, data revealed differentially regulated analytes across tissues, such as CCL11, CXCL5, and IL-10 in the brain, IL-33/IL-33R in the liver, and VEGF-a and IL-5 in the lungs. Overall, this study provides an overview of the immune dynamics of soluble mediators in congenital CMV.
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Infecções por Citomegalovirus , Muromegalovirus , Animais , Humanos , Camundongos , Citocinas , Encéfalo , Fator de Necrose Tumoral alfaRESUMO
Meningococcal glycoconjugate vaccines sourced from capsular polysaccharides (CPSs) of pathogenic Neisseria meningitidis strains are well-established measures to prevent meningococcal disease. However, the exact structural factors responsible for antibody recognition are not known. CPSs of Neisseria meningitidis serogroups Y and W differ by a single stereochemical center, yet they evoke specific immune responses. Herein, we developed specific monoclonal antibodies (mAbs) targeting serogroups C, Y, and W and evaluated their ability to kill bacteria. We then used these mAbs to dissect structural elements responsible for carbohydrate-protein interactions. First, Men oligosaccharides were screened against the mAbs using ELISA to select putative lengths representing the minimal antigenic determinant. Next, molecular interaction features between the mAbs and serogroup-specific sugar fragments were elucidated using STD-NMR. Moreover, X-ray diffraction data with the anti-MenW CPS mAb enabled the elucidation of the sugar-antibody binding mode. Our findings revealed common traits in the epitopes of all three sialylated serogroups. The minimal binding epitopes typically comprise five to six repeating units. Moreover, the O-acetylation of the neuraminic acid moieties was fundamental for mAb binding. These insights hold promise for the rational design of optimized meningococcal oligosaccharides, opening new avenues for novel production methods, including chemical or enzymatic approaches.
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Anticorpos Monoclonais , Vacinas Meningocócicas , Neisseria meningitidis , Polissacarídeos Bacterianos , Sorogrupo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/química , Neisseria meningitidis/imunologia , Neisseria meningitidis/química , Vacinas Meningocócicas/imunologia , Vacinas Meningocócicas/química , Polissacarídeos Bacterianos/imunologia , Polissacarídeos Bacterianos/química , Anticorpos Antibacterianos/imunologia , Epitopos/imunologia , Epitopos/química , Animais , Camundongos , Humanos , Cápsulas Bacterianas/imunologia , Cápsulas Bacterianas/química , Formação de Anticorpos/imunologiaRESUMO
Extensive reorganization of infected cells and the formation of large structures known as the nuclear replication compartment (RC) and cytoplasmic assembly compartment (AC) is a hallmark of beta-herpesvirus infection. These restructurings rely on extensive compartmentalization of the processes that make up the virus manufacturing chain. Compartmentalization of the nuclear processes during murine cytomegalovirus (MCMV) infection is not well described. In this study, we visualized five viral proteins (pIE1, pE1, pM25, pm48.2, and pM57) and replicated viral DNA to reveal the nuclear events during MCMV infection. As expected, these events can be matched with those described for other beta and alpha herpesviruses and contribute to the overall picture of herpesvirus assembly. Imaging showed that four viral proteins (pE1, pM25, pm48.2, and pM57) and replicated viral DNA condense in the nucleus into membraneless assemblies (MLAs) that undergo a maturation sequence to form the RC. One of these proteins (pM25), which is also expressed in a cytoplasmic form (pM25l), showed similar MLAs in the AC. Bioinformatics tools for predicting biomolecular condensates showed that four of the five proteins had a high propensity for liquid-liquid phase separation (LLPS), suggesting that LLPS may be a mechanism for compartmentalization within RC and AC. Examination of the physical properties of MLAs formed during the early phase of infection by 1,6-hexanediol treatment in vivo revealed liquid-like properties of pE1 MLAs and more solid-like properties of pM25 MLAs, indicating heterogeneity of mechanisms in the formation of virus-induced MLAs. Analysis of the five viral proteins and replicated viral DNA shows that the maturation sequence of RC and AC is not completed in many cells, suggesting that virus production and release is carried out by a rather limited number of cells. This study thus lays the groundwork for further investigation of the replication cycle of beta-herpesviruses, and the results should be incorporated into plans for high-throughput and single-cell analytic approaches.
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Infecções por Citomegalovirus , Muromegalovirus , Vírus , Animais , Camundongos , Muromegalovirus/metabolismo , DNA Viral/genética , Núcleo Celular/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírus/metabolismoRESUMO
Infections in early life can elicit substantially different immune responses and pathogenesis than infections in adulthood. Here, we investigate the consequences of murine cytomegalovirus infection in newborn mice on NK cells. We show that infection severely compromised NK cell maturation and functionality in newborns. This effect was not due to compromised virus control. Inflammatory responses to infection dysregulated the expression of major transcription factors governing NK cell fate, such as Eomes, resulting in impaired NK cell function. Most prominently, NK cells from perinatally infected mice have a diminished ability to produce IFN-γ due to the downregulation of long non-coding RNA Ifng-as1 expression. Moreover, the bone marrow's capacity to efficiently generate new NK cells is reduced, explaining the prolonged negative effects of perinatal infection on NK cells. This study demonstrates that viral infections in early life can profoundly impact NK cell biology, including long-lasting impairment in NK cell functionality.
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Infecções por Citomegalovirus , Muromegalovirus , Camundongos , Animais , Células Matadoras Naturais , Infecções por Citomegalovirus/genéticaRESUMO
Bovine mastitis is the most frequent disease on dairy farms, which leads to a decrease in the health welfare of the animals and great economic losses. This study was aimed at determining the quantitative variations in the milk proteome caused by natural infection by Staphylococcus and Streptococcus species in order to gain further understanding of any discrepancies in pathophysiology and host immune responses, independent of the mastitis level. After identification of Staphylococcus (N = 51) and Streptococcus (N = 67) spp., tandem mass tag (TMT)-labeled quantitative proteomic and liquid chromatography-mass spectrometry (LC-MS/MS) techniques on a modular Ultimate 3000 RSLCnano system coupled to a Q Exactive Plus was applied on aseptically sampled milk from Holstein cows. Proteome Discoverer was used for protein identification and quantitation through the SEQUEST algorithm. Statistical analysis employing R was used to identify differentially abundant proteins between the groups. Protein classes, functions and functional-association networks were determined using the PANTHER and STRING tools and pathway over-representation using the REACTOME. In total, 156 master bovine proteins were identified (two unique peptides, p < 0.05 and FDR < 0.001), and 20 proteins showed significantly discrepant abundance between the genera (p < 0.05 and FDR < 0.5). The most discriminatory proteins per group were odorant-binding protein (higher in staphylococci) and fibrinogen beta chain protein (higher in streptococci). The receiver operating characteristic (ROC) curve showed that protein kinase C-binding protein NELL2, thrombospondin-1, and complement factor I have diagnostic potential for differentiating staphylococci and streptococci intramammary infection and inflammation. Improved understanding of the host response mechanisms and recognition of potential biomarkers of specific-pathogen mastitis, which may aid prompt diagnosis for control implementation, are potential benefits of this study.
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This article describes procedures for infecting adult mice with murine cytomegalovirus (MCMV) and for infecting newborn mice to model congenital CMV infection. Methods are included for propagating MCMV in cell cultures and preparing a more virulent form of MCMV from the salivary glands of infected mice. A plaque assay is provided for determining MCMV titers of infected tissues or virus stocks. Also, methods are described for preparing the murine embryonic fibroblasts used for propagating MCMV, and for the plaque assay. © 2022 Wiley Periodicals LLC.
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Infecções por Citomegalovirus , Muromegalovirus , Animais , Modelos Animais de Doenças , Camundongos , Glândulas SalivaresRESUMO
Herpes simplex virus 1 (HSV-1) expresses a large number of miRNAs, and their function is still not completely understood. In addition, HSV-1 has been found to deregulate host miRNAs, which adds to the complexity of the regulation of efficient virus replication. In this study, we comprehensively addressed the deregulation of host miRNAs by massive-parallel sequencing. We found that only miRNAs expressed from a single cluster, miR-183/96/182, are reproducibly deregulated during productive infection. These miRNAs are predicted to regulate a great number of potential targets involved in different cellular processes and have only 33 shared targets. Among these, members of the FoxO family of proteins were identified as potential targets for all three miRNAs. However, our study shows that the upregulated miRNAs do not affect the expression of FoxO proteins, moreover, these proteins were upregulated in HSV-1 infection. Furthermore, we show that the individual FoxO proteins are not required for efficient HSV-1 replication. Taken together, our results indicate a complex and redundant response of infected cells to the virus infection that is efficiently inhibited by the virus.
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Herpes Simples , Herpesvirus Humano 1 , MicroRNAs , Herpes Simples/genética , Herpesvirus Humano 1/fisiologia , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Regulação para Cima , Replicação ViralRESUMO
Human cytomegalovirus (HCMV) is a leading viral cause of congenital infections in the central nervous system (CNS) and may result in severe long-term sequelae. High rates of sequelae following congenital HCMV infection and insufficient antiviral therapy in the perinatal period makes the development of an HCMV-specific vaccine a high priority of modern medicine. Due to the species specificity of HCMV, animal models are frequently used to study CMV pathogenesis. Studies of murine cytomegalovirus (MCMV) infections of adult mice have played a significant role as a model of CMV biology and pathogenesis, while MCMV infection of newborn mice has been successfully used as a model of perinatal CMV infection. Newborn mice infected with MCMV have high levels of viremia during which the virus establishes a productive infection in most organs, coupled with a robust inflammatory response. Productive infection in the brain parenchyma during early postnatal period leads to an extensive nonnecrotizing multifocal widespread encephalitis characterized by infiltration of components of both innate and adaptive immunity. As a result, impairment in postnatal development of mouse cerebellum leads to long-term motor and sensor disabilities. This chapter summarizes current findings of rodent models of perinatal CMV infection and describes methods for analysis of perinatal MCMV infection in newborn mice.
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Citomegalovirus/imunologia , Modelos Animais de Doenças , Animais , Animais Recém-Nascidos , Encéfalo/imunologia , Sistema Nervoso Central/virologia , Citomegalovirus/metabolismo , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/imunologia , Encefalite , Doenças Fetais , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Muromegalovirus/imunologia , Cultura Primária de CélulasRESUMO
Viral vectors have emerged as a promising alternative to classical vaccines due to their great potential for induction of a potent cellular and humoral immunity. Cytomegalovirus (CMV) is an attractive vaccine vector due to its large genome with many non-essential immunoregulatory genes that can be easily manipulated to modify the immune response. CMV generates a strong antigen-specific CD8 T cell response with a gradual accumulation of these cells in the process called memory inflation. In our previous work, we have constructed a mouse CMV vector expressing NKG2D ligand RAE-1γ in place of its viral inhibitor m152 (RAE-1γMCMV), which proved to be highly attenuated in vivo. Despite attenuation, RAE-1γMCMV induced a substantially stronger CD8 T cell response to vectored antigen than the control vector and provided superior protection against bacterial and tumor challenge. In the present study, we confirmed the enhanced protective capacity of RAE-1γMCMV as a tumor vaccine vector and determined the phenotypical and functional characteristics of memory CD8 T cells induced by the RAE-1γ expressing MCMV. RNAseq data revealed higher transcription of numerous genes associated with effector-like CD8 T cell phenotype in RAE-1γMCMV immunized mice. CD8 T cells primed with RAE-1γMCMV were enriched in TCF1 negative population, with higher expression of KLRG1 and lower expression of CD127, CD27, and Eomes. These phenotypical differences were associated with distinct functional features as cells primed with RAE-1γMCMV showed inferior cytokine-producing abilities but comparable cytotoxic potential. After adoptive transfer into naive hosts, OT-1 cells induced with both RAE-1γMCMV and the control vector were equally efficient in rejecting established tumors, suggesting the context of latent infection and cell numbers as important determinants of enhanced anti-tumor response following RAE-1γMCMV vaccination. Overall, our results shed new light on the phenotypical and functional distinctness of memory CD8 T cells induced with CMV vector expressing cellular ligand for the NKG2D receptor.
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Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vacinas contra Citomegalovirus/imunologia , Memória Imunológica , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Fenótipo , Animais , Vacinas Anticâncer/imunologia , Biologia Computacional/métodos , Citomegalovirus/imunologia , Citotoxicidade Imunológica , Perfilação da Expressão Gênica , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/prevenção & controle , Imunofenotipagem , Ativação Linfocitária/imunologia , Camundongos , Muromegalovirus/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , TranscriptomaRESUMO
During COVID-19 pandemics, the availability of testing has often been a limiting factor during patient admissions into the hospital. To circumvent this problem, we adapted an existing diagnostic assay, Seegene Allplex SARS-CoV-2, into a point-of-care-style direct qPCR (POC dqPCR) assay and implemented it in the Emergency Department of Clinical Hospital Center Rijeka, Croatia. In a 4-month analysis, we tested over 10,000 patients and demonstrated that POC-dqPCR is robust and reliable and can be successfully implemented in emergency departments and similar near-patient settings and can be performed by medical personnel with little prior experience in qPCR.
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Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Serviço Hospitalar de Emergência , Testes Imediatos , SARS-CoV-2/isolamento & purificação , COVID-19/epidemiologia , Croácia/epidemiologia , Humanos , RNA Viral/genética , Reprodutibilidade dos Testes , SARS-CoV-2/genética , Sensibilidade e EspecificidadeRESUMO
Congenital human cytomegalovirus (cHCMV) infection of the brain is associated with a wide range of neurocognitive sequelae. Using infection of newborn mice with mouse cytomegalovirus (MCMV) as a reliable model that recapitulates many aspects of cHCMV infection, including disseminated infection, CNS infection, altered neurodevelopment, and sensorineural hearing loss, we have previously shown that mitigation of inflammation prevented alterations in cerebellar development, suggesting that host inflammatory factors are key drivers of neurodevelopmental defects. Here, we show that MCMV infection causes a dramatic increase in the expression of the microglia-derived chemokines CXCL9/CXCL10, which recruit NK and ILC1 cells into the brain in a CXCR3-dependent manner. Surprisingly, brain-infiltrating innate immune cells not only were unable to control virus infection in the brain but also orchestrated pathological inflammatory responses, which lead to delays in cerebellar morphogenesis. Our results identify NK and ILC1 cells as the major mediators of immunopathology in response to virus infection in the developing CNS, which can be prevented by anti-IFN-γ antibodies.
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Encéfalo/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Inflamação/imunologia , Células Matadoras Naturais/imunologia , Linfócitos/imunologia , Animais , Animais Recém-Nascidos , Encéfalo/patologia , Encéfalo/virologia , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Quimiocina CXCL10/metabolismo , Quimiocina CXCL9/genética , Quimiocina CXCL9/imunologia , Quimiocina CXCL9/metabolismo , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/virologia , Regulação da Expressão Gênica/imunologia , Humanos , Imunidade Inata/imunologia , Inflamação/genética , Inflamação/virologia , Células Matadoras Naturais/metabolismo , Linfócitos/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/imunologia , Microglia/metabolismo , Microglia/virologia , Receptores CXCR3/genética , Receptores CXCR3/imunologia , Receptores CXCR3/metabolismoRESUMO
Despite the considerable progress toward the eradication of meningococcal disease with the introduction of glycoconjugate vaccines, previously unremarkable serogroup X has emerged in recent years, recording several outbreaks throughout the African continent. Different serogroup X polysaccharide-based vaccines have been tested in preclinical trials, establishing the principles for further improvement. To elucidate the antigenic determinants of the MenX capsular polysaccharide, we generated a monoclonal antibody, and its bactericidal nature was confirmed using the rabbit serum bactericidal assay. The antibody was tested by the inhibition enzyme-linked immunosorbent assay and surface plasmon resonance against a set of oligosaccharide fragments of different lengths. The epitope was shown to be contained within five to six α-(1-4) phosphodiester mannosamine repeating units. The molecular interactions between the protective monoclonal antibody and the MenX capsular polysaccharide fragment were further detailed at the atomic level by saturation transfer difference nuclear magnetic resonance (NMR) spectroscopy. The NMR results were used for validation of the in silico docking analysis between the X-ray crystal structure of the antibody (Fab fragment) and the modeled hexamer oligosaccharide. The antibody recognizes the MenX fragment by binding all six repeating units of the oligosaccharide via hydrogen bonding, salt bridges, and hydrophobic interactions. In vivo studies demonstrated that conjugates containing five to six repeating units can produce high functional antibody levels. These results provide an insight into the molecular basis of MenX vaccine-induced protection and highlight the requirements for the epitope-based vaccine design.
RESUMO
Palindromic and quasi-palindromic sequences are important DNA motifs found in various cis-acting genetic elements, but are also known to provoke different types of genetic alterations. The instability of such motifs is clearly size-related and depends on their potential to adopt secondary structures known as hairpins and cruciforms. Here we studied the influence of palindrome size on recombination between two directly repeated copies of the yeast CYC1 gene leading to the loss of the intervening sequence ("pop-out" recombination). We show that palindromes inserted either within one copy or between the two copies of the CYC1 gene become recombinogenic only when they attain a certain critical size and we estimate this critical size to be about 70 bp. With the longest palindrome used in this study (150 bp) we observed a more than 20-fold increase in the pop-out recombination. In the sae2/com1 mutant the palindrome-stimulated recombination was completely abolished. Suppression of palindrome recombinogenicity may be crucial for the maintenance of genetic stability in organisms containing a significant number of large palindromes in their genomes, like humans.