RESUMO
Cytomegaloviruses (CMVs) transmit via chronic shedding from the salivary glands. How this relates to the broad cell tropism they exhibit in vitro is unclear. Human CMV (HCMV) infection presents only after salivary gland infection is established. Murine CMV (MCMV) is therefore useful to analyse early infection events. It reaches the salivary glands via infected myeloid cells. Three adjacent spliced genes designated as m131/129 (MCK-2), sgg1 and sgg1.1, positional homologues of the HCMV UL128/130/131 tropism determinants, are implicated. We show that a sgg1 null mutant is defective in infected myeloid cell entry into the salivary glands, a phenotype distinct from MCMV lacking MCK-2. These data point to a complex, multi-step process of salivary gland colonization.
Assuntos
Muromegalovirus , Glândulas Salivares , Animais , Glândulas Salivares/virologia , Muromegalovirus/genética , Muromegalovirus/fisiologia , Camundongos , Tropismo Viral , Células Mieloides/virologia , Células Mieloides/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Infecções por Herpesviridae/virologia , Quimiocinas CCRESUMO
Current vaccines against COVID-19 elicit immune responses that are overall strong but wane rapidly. As a consequence, the necessary booster shots have contributed to vaccine fatigue. Hence, vaccines that would provide lasting protection against COVID-19 are needed, but are still unavailable. Cytomegaloviruses (CMVs) elicit lasting and uniquely strong immune responses. Used as vaccine vectors, they may be attractive tools that obviate the need for boosters. Therefore, we tested the murine CMV (MCMV) as a vaccine vector against COVID-19 in relevant preclinical models of immunization and challenge. We have previously developed a recombinant MCMV vaccine vector expressing the spike protein of the ancestral SARS-CoV-2 (MCMVS). In this study, we show that the MCMVS elicits a robust and lasting protection in young and aged mice. Notably, spike-specific humoral and cellular immunity was not only maintained but also even increased over a period of at least 6 months. During that time, antibody avidity continuously increased and expanded in breadth, resulting in neutralization of genetically distant variants, like Omicron BA.1. A single dose of MCMVS conferred rapid virus clearance upon challenge. Moreover, MCMVS vaccination controlled two variants of concern (VOCs), the Beta (B.1.135) and the Omicron (BA.1) variants. Thus, CMV vectors provide unique advantages over other vaccine technologies, eliciting broadly reactive and long-lasting immune responses against COVID-19.
Assuntos
Anticorpos Antivirais , Vacinas contra COVID-19 , COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Camundongos , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Muromegalovirus/imunologia , Muromegalovirus/genética , Feminino , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Camundongos Endogâmicos BALB C , Humanos , Vetores Genéticos , Imunidade Celular , Imunidade Humoral , Modelos Animais de DoençasRESUMO
There are no licensed vaccines for human cytomegalovirus (HCMV), and current antiviral drugs that target viral proteins are toxic and prone to resistance. Targeting host pathways essential for virus replication provides an alternate strategy that may reduce opportunities for drug resistance to occur. Oxidative stress is triggered by numerous viruses including HCMV. Peroxynitrite is a reactive nitrogen species that is formed during oxidative stress. Herein, we identified that HCMV rapidly induces the generation of intracellular peroxynitrite upon infection in a manner partially dependent upon xanthine oxidase generation. Peroxynitrite promoted HCMV infection in both cell-free and cell-associated infection systems in multiple cell types. Inhibiting peroxynitrite within the first 24 hours of infection prevented HCMV replication and peroxynitrite promoted cell entry and pp65 translocation into the host cell nuclei. Furthermore, using the murine cytomegalovirus model, we demonstrated that antagonizing peroxynitrite significantly reduces cytomegalovirus replication and pathogenesis in vivo. Overall, our study highlights a proviral role for peroxynitrite in CMV infection and implies that RNS and/or the mechanisms that induce their production could be targeted as a novel strategy to inhibit HCMV infection. IMPORTANCE: Human cytomegalovirus (HCMV) causes significant disease in individuals with impaired or immature immune systems, such as transplant patients and after congenital infection. Antiviral drugs that target the virus directly are toxic and are susceptible to antiviral drug resistance due to virus mutations. An alternate strategy is to target processes within host cells that are required by the virus for replication. Herein, we show that HCMV infection triggers a highly reactive molecule, peroxynitrite, during the initial stages of infection. Peroxynitrite was required for the initial entry of the virus into the cell and promotes virus replication in multiple cell types, suggesting a broad pro-viral function. Importantly, targeting peroxynitrite dramatically inhibited cytomegalovirus replication in cells in the laboratory and in mice, suggesting that therapeutic targeting of this molecule and/or the cellular functions it regulates could represent a novel strategy to inhibit HCMV infection.
Assuntos
Infecções por Citomegalovirus , Citomegalovirus , Modelos Animais de Doenças , Ácido Peroxinitroso , Internalização do Vírus , Replicação Viral , Ácido Peroxinitroso/metabolismo , Ácido Peroxinitroso/farmacologia , Animais , Camundongos , Citomegalovirus/fisiologia , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/genética , Internalização do Vírus/efeitos dos fármacos , Humanos , Infecções por Citomegalovirus/virologia , Replicação Viral/efeitos dos fármacos , Muromegalovirus/fisiologia , Muromegalovirus/efeitos dos fármacos , Linhagem Celular , Estresse OxidativoRESUMO
The involvement of γδ TCR-bearing lymphocytes in immunological memory has gained increasing interest due to their functional duality between adaptive and innate immunity. γδ T effector memory (TEM) and central memory (TCM) subsets have been identified, but their respective roles in memory responses are poorly understood. In the present study, we used subsequent mouse cytomegalovirus (MCMV) infections of αß T cell deficient mice in order to analyze the memory potential of γδ T cells. As for CMV-specific αß T cells, MCMV induced the accumulation of cytolytic, KLRG1+CX3CR1+ γδ TEM that principally localized in infected organ vasculature. Typifying T cell memory, γδ T cell expansion in organs and blood was higher after secondary viral challenge than after primary infection. Viral control upon MCMV reinfection was prevented when masking γδ T-cell receptor, and was associated with a preferential amplification of private and unfocused TCR δ chain repertoire composed of a combination of clonotypes expanded post-primary infection and, more unexpectedly, of novel expanded clonotypes. Finally, long-term-primed γδ TCM cells, but not γδ TEM cells, protected T cell-deficient hosts against MCMV-induced death upon adoptive transfer, probably through their ability to survive and to generate TEM in the recipient host. This better survival potential of TCM cells was confirmed by a detailed scRNASeq analysis of the two γδ T cell memory subsets which also revealed their similarity to classically adaptive αß CD8 T cells. Overall, our study uncovered memory properties of long-lived TCM γδ T cells that confer protection in a chronic infection, highlighting the interest of this T cell subset in vaccination approaches.
Assuntos
Infecções por Herpesviridae , Memória Imunológica , Células T de Memória , Muromegalovirus , Receptores de Antígenos de Linfócitos T gama-delta , Animais , Camundongos , Muromegalovirus/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Infecções por Herpesviridae/imunologia , Memória Imunológica/imunologia , Células T de Memória/imunologia , Reinfecção/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Citomegalovirus/imunologiaRESUMO
Natural killer (NK) cells function by eliminating virus-infected or tumor cells. Here we identified an NK-lineage-biased progenitor population, referred to as early NK progenitors (ENKPs), which developed into NK cells independently of common precursors for innate lymphoid cells (ILCPs). ENKP-derived NK cells (ENKP_NK cells) and ILCP-derived NK cells (ILCP_NK cells) were transcriptionally different. We devised combinations of surface markers that identified highly enriched ENKP_NK and ILCP_NK cell populations in wild-type mice. Furthermore, Ly49H+ NK cells that responded to mouse cytomegalovirus infection primarily developed from ENKPs, whereas ILCP_NK cells were better IFNγ producers after infection with Salmonella and herpes simplex virus. Human CD56dim and CD56bright NK cells were transcriptionally similar to ENKP_NK cells and ILCP_NK cells, respectively. Our findings establish the existence of two pathways of NK cell development that generate functionally distinct NK cell subsets in mice and further suggest these pathways may be conserved in humans.
Assuntos
Diferenciação Celular , Células Matadoras Naturais , Células Matadoras Naturais/imunologia , Animais , Camundongos , Humanos , Diferenciação Celular/imunologia , Camundongos Endogâmicos C57BL , Imunidade Inata , Antígeno CD56/metabolismo , Muromegalovirus/imunologia , Linhagem da Célula/imunologia , Interferon gama/metabolismo , Interferon gama/imunologia , Células Progenitoras Linfoides/metabolismo , Células Progenitoras Linfoides/citologia , Células Progenitoras Linfoides/imunologia , Camundongos Knockout , Células CultivadasRESUMO
Human cytomegalovirus (HCMV) infection in infants infected in utero can lead to a variety of neurodevelopmental disorders. However, mechanisms underlying altered neurodevelopment in infected infants remain poorly understood. We have previously described a murine model of congenital HCMV infection in which murine CMV (MCMV) spreads hematogenously and establishes a focal infection in all regions of the brain of newborn mice, including the cerebellum. Infection resulted in disruption of cerebellar cortical development characterized by reduced cerebellar size and foliation. This disruption was associated with altered cell cycle progression of the granule cell precursors (GCPs), which are the progenitors that give rise to granule cells (GCs), the most abundant neurons in the cerebellum. In the current study, we have demonstrated that MCMV infection leads to prolonged GCP cell cycle, premature exit from the cell cycle, and reduced numbers of GCs resulting in cerebellar hypoplasia. Treatment with TNF-α neutralizing antibody partially normalized the cell cycle alterations of GCPs and altered cerebellar morphogenesis induced by MCMV infection. Collectively, our results argue that virus-induced inflammation altered the cell cycle of GCPs resulting in a reduced numbers of GCs and cerebellar cortical hypoplasia, thus providing a potential mechanism for altered neurodevelopment in fetuses infected with HCMV.
Assuntos
Ciclo Celular , Cerebelo , Infecções por Citomegalovirus , Modelos Animais de Doenças , Animais , Infecções por Citomegalovirus/virologia , Infecções por Citomegalovirus/patologia , Camundongos , Cerebelo/virologia , Cerebelo/patologia , Cerebelo/crescimento & desenvolvimento , Cerebelo/anormalidades , Feminino , Citomegalovirus , Células-Tronco Neurais/virologia , Muromegalovirus/fisiologia , Animais Recém-Nascidos , Humanos , Neurônios/virologia , Fator de Necrose Tumoral alfa/metabolismo , Deficiências do Desenvolvimento , Malformações do Sistema NervosoRESUMO
Human cytomegalovirus is a ubiquitous herpesvirus that, while latent in most individuals, poses a great risk to immunocompromised patients. In contrast to directly acting traditional antiviral drugs, such as ganciclovir, we aim to emulate a physiological infection control using T cells. For this, we constructed several bispecific T-cell engager (BiTE) constructs targeting different viral glycoproteins of the murine cytomegalovirus and evaluated them in vitro for their efficacy. To isolate the target specific effect without viral immune evasion, we established stable reporter cell lines expressing the viral target glycoprotein B, and the glycoprotein complexes gN-gM and gH-gL, as well as nano-luciferase (nLuc). First, we evaluated binding capacities using flow cytometry and established killing assays, measuring nLuc-release upon cell lysis. All BiTE constructs proved to be functional mediators for T-cell recruitment and will allow a proof of concept for this treatment option. This might pave the way for strikingly safer immunosuppression in vulnerable patient groups.
Assuntos
Muromegalovirus , Linfócitos T , Animais , Linfócitos T/imunologia , Camundongos , Muromegalovirus/imunologia , Muromegalovirus/fisiologia , Humanos , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/imunologia , Linhagem Celular , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
The tegument protein pp150 of Human Cytomegalovirus (HCMV) is known to be essential for the final stages of virus maturation and mediates its functions by interacting with capsid proteins. Our laboratory has previously identified the critical regions in pp150 important for pp150-capsid interactions and designed peptides similar in sequence to these regions, with a goal to competitively inhibit capsid maturation. Treatment with a specific peptide (PepCR2 or P10) targeted to pp150 conserved region 2 led to a significant reduction in murine CMV (MCMV) growth in cell culture, paving the way for in vivo testing in a mouse model of CMV infection. However, the general pharmacokinetic parameters of peptides, including rapid degradation and limited tissue and cell membrane permeability, pose a challenge to their successful use in vivo. Therefore, we designed a biopolymer-stabilized elastin-like polypeptide (ELP) fusion construct (ELP-P10) to enhance the bioavailability of P10. Antiviral efficacy and cytotoxic effects of ELP-P10 were studied in cell culture, and pharmacokinetics, biodistribution, and antiviral efficacy were studied in a mouse model of CMV infection. ELP-P10 maintained significant antiviral activity in cell culture, and this conjugation significantly enhanced P10 bioavailability in mouse tissues. The fluorescently labeled ELP-P10 accumulated to higher levels in mouse liver and kidneys as compared to the unconjugated P10. Moreover, viral titers from vital organs of MCMV-infected mice indicated a significant reduction of virus load upon ELP-P10 treatment. Therefore, ELP-P10 has the potential to be developed into an effective antiviral against CMV infection.
Assuntos
Antivirais , Infecções por Citomegalovirus , Elastina , Muromegalovirus , Peptídeos , Fosfoproteínas , Proteínas da Matriz Viral , Animais , Camundongos , Antivirais/farmacologia , Antivirais/farmacocinética , Antivirais/química , Capsídeo/metabolismo , Capsídeo/efeitos dos fármacos , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/química , Citomegalovirus/efeitos dos fármacos , Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Citomegalovirus/virologia , Modelos Animais de Doenças , Elastina/química , Elastina/metabolismo , Polipeptídeos Semelhantes à Elastina , Muromegalovirus/efeitos dos fármacos , Peptídeos/farmacologia , Peptídeos/química , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/farmacocinéticaRESUMO
Chemokines guide immune cells during their response against pathogens and tumors. Various techniques exist to determine chemokine production, but none to identify cells that directly sense chemokines in vivo. We have generated CCL3-EASER (ErAse, SEnd, Receive) mice that simultaneously report for Ccl3 transcription and translation, allow identifying Ccl3-sensing cells, and permit inducible deletion of Ccl3-producing cells. We infected these mice with murine cytomegalovirus (mCMV), where Ccl3 and NK cells are critical defense mediators. We found that NK cells transcribed Ccl3 already in homeostasis, but Ccl3 translation required type I interferon signaling in infected organs during early infection. NK cells were both the principal Ccl3 producers and sensors of Ccl3, indicating auto/paracrine communication that amplified NK cell response, and this was essential for the early defense against mCMV. CCL3-EASER mice represent the prototype of a new class of dual fluorescence reporter mice for analyzing cellular communication via chemokines, which may be applied also to other chemokines and disease models.
Assuntos
Comunicação Celular , Quimiocina CCL3 , Modelos Animais , Biossíntese de Proteínas , Transcrição Gênica , Animais , Camundongos , Comunicação Celular/imunologia , Quimiocina CCL3/genética , Quimiocina CCL3/imunologia , Técnicas de Introdução de Genes , Camundongos Transgênicos , Muromegalovirus , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/imunologia , Transcrição Gênica/imunologia , Células Matadoras Naturais/imunologia , Interferon beta/farmacologia , Infecções por Herpesviridae/imunologiaRESUMO
Reactivation and infection with cytomegalovirus (CMV) are frequently observed in recipients of solid organ transplants, bone marrow transplants, and individuals with HIV infection. This presents an increasing risk of allograft rejection, opportunistic infection, graft failure, and patient mortality. Among immunocompromised hosts, interstitial pneumonia is the most critical clinical manifestation of CMV infection. Recent studies have demonstrated the potential therapeutic benefits of exosomes derived from mesenchymal stem cells (MSC-exos) in preclinical models of acute lung injury, including pneumonia, ARDS, and sepsis. However, the role of MSC-exos in the pathogenesis of infectious viral diseases, such as CMV pneumonia, remains unclear. In a mouse model of murine CMV-induced pneumonia, we observed that intravenous administration of mouse MSC (mMSC)-exos reduced lung damage, decreased the hyperinflammatory response, and shifted macrophage polarization from the M1 to the M2 phenotype. Treatment with mMSC-exos also significantly reduced the infiltration of inflammatory cells and pulmonary fibrosis. Furthermore, in vitro studies revealed that mMSC-exos reversed the hyperinflammatory phenotype of bone marrow-derived macrophages infected with murine CMV. Mechanistically, mMSC-exos treatment decreased activation of the NF-κB/NLRP3 signaling pathway both in vivo and in vitro. In summary, our findings indicate that mMSC-exo treatment is effective in severe CMV pneumonia by reducing lung inflammation and fibrosis through the NF-κB/NLRP3 signaling pathway, thus providing promising therapeutic potential for clinical CMV infection.
Assuntos
Modelos Animais de Doenças , Exossomos , Células-Tronco Mesenquimais , Muromegalovirus , NF-kappa B , Proteína 3 que Contém Domínio de Pirina da Família NLR , Transdução de Sinais , Animais , Exossomos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , NF-kappa B/metabolismo , Muromegalovirus/fisiologia , Camundongos Endogâmicos C57BL , Macrófagos/imunologia , Infecções por Citomegalovirus/terapia , Infecções por Citomegalovirus/virologia , Pulmão/virologia , Pulmão/patologia , Pneumonia Viral/terapia , Pneumonia Viral/virologia , Infecções por Herpesviridae/terapia , Infecções por Herpesviridae/virologia , Infecções por Herpesviridae/imunologia , Pneumonia/terapia , Pneumonia/virologiaRESUMO
Human cytomegalovirus (hCMV) is a ubiquitous facultative pathogen, which establishes a characteristic latent and reactivating lifelong infection in immunocompetent hosts. Murine CMV (mCMV) infection is widely used as an experimental model of hCMV infection, employed to investigate the causal nature and extent of CMV's contribution to inflammatory, immunological, and health disturbances in humans. Therefore, mimicking natural human infection in mice would be advantageous to hCMV research. To assess the role of route and age at infection in modeling hCMV in mice, we infected prepubescent and young sexually mature C57BL/6 (B6) mice intranasally (i.n., a likely physiological route in humans) and intraperitoneally (i.p., a frequently used experimental route, possibly akin to transplant-mediated infection). In our hands, both routes led to comparable early viral loads and tissue spreads. However, they yielded differential profiles of innate and adaptive systemic immune activation. Specifically, the younger, prepubescent mice exhibited the strongest natural killer cell activation in the blood in response to i.p. infection. Further, the i.p. infected animals (particularly those infected at 12 weeks) exhibited larger anti-mCMV IgG and greater expansion of circulating CD8+ T cells specific for both acute (non-inflationary) and latent phase (inflationary) mCMV epitopes. By contrast, tissue immune responses were comparable between i.n. and i.p. groups. Our results illustrate a distinction in the bloodborne immune response profiles across infection routes and ages and are discussed in light of physiological parameters of interaction between CMV, immunity, inflammation, and health over the lifespan. IMPORTANCE: The majority of experiments modeling human cytomegalovirus (hCMV) infection in mice have been carried out using intraperitoneal infection in sexually mature adult mice, which stands in contrast to the large number of humans being infected with human CMV at a young age, most likely via bodily fluids through the nasopharyngeal/oral route. This study examined the impact of the choice of age and route of infection in modeling CMV infection in mice. By comparing young, prepubescent to older sexually mature counterparts, infected either via the intranasal or intraperitoneal route, we discovered substantial differences in deployment and response intensity of different arms of the immune system in systemic control of the virus; tissue responses, by contrast, appeared similar between ages and infection routes.
Assuntos
Imunidade Adaptativa , Infecções por Citomegalovirus , Imunidade Inata , Muromegalovirus , Animais , Feminino , Humanos , Camundongos , Fatores Etários , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Modelos Animais de Doenças , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Células Matadoras Naturais/imunologia , Camundongos Endogâmicos C57BL , Muromegalovirus/imunologia , Carga ViralRESUMO
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.
Assuntos
Infecções por Citomegalovirus , Muromegalovirus , Animais , Humanos , Camundongos , Citocinas , Encéfalo , Fator de Necrose Tumoral alfaRESUMO
For successful viral propagation within infected cells, the virus needs to overcome the cellular integrated stress response (ISR), triggered during viral infection, which, in turn, inhibits general protein translation. This paper reports a tactic employed by viruses to suppress the ISR by upregulating host cell polyribonucleotide nucleotidyltransferase 1 (PNPT1). The propagation of adenovirus, murine cytomegalovirus and hepatovirus within their respective host cells induces PNPT1 expression. Notably, when PNPT1 is knocked down, the propagation of all three viruses is prevented. Mechanistically, the inhibition of PNPT1 facilitates the relocation of mitochondrial double-stranded RNAs (mt-dsRNAs) to the cytoplasm, where they activate RNA-activated protein kinase (PKR). This activation leads to eukaryotic initiation factor 2α (eIF2α) phosphorylation, resulting in the suppression of translation. Furthermore, by scrutinizing the PNPT1 recognition element and screening 17,728 drugs and bioactive compounds approved by the US Food and Drug Administration, lanatoside C was identified as a potent PNPT1 inhibitor. This compound impedes the propagation of adenovirus, murine cytomegalovirus and hepatovirus, and suppresses production of the severe acute respiratory syndrome coronavirus-2 spike protein. These discoveries shed light on a novel strategy to impede pan-viral propagation by activating the host cell mt-dsRNA-PKR-eIF2α signalling axis.
Assuntos
eIF-2 Quinase , Humanos , Animais , eIF-2 Quinase/metabolismo , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/genética , Antivirais/farmacologia , Muromegalovirus/fisiologia , Muromegalovirus/efeitos dos fármacos , Camundongos , Fator de Iniciação 2 em Eucariotos/metabolismo , Replicação Viral/efeitos dos fármacos , RNA de Cadeia Dupla/genética , Adenoviridae/genética , Adenoviridae/efeitos dos fármacos , Fosforilação , SARS-CoV-2/efeitos dos fármacosRESUMO
The use of nanoparticles as a delivery system for a specific antigen could solve many limitations of mucosal vaccine applications, such as low immunogenicity, or antigen protection and stabilization. In this study, we tested the ability of nasally administered chitosan nanoparticles loaded with glycoprotein B of murine cytomegalovirus to induce an immune response in an animal model. The choice of chitosan nanoparticle type was made by in vitro evaluation of sorption efficiency and antigen release. Three types of chitosan nanoparticles were prepared: crosslinked with tripolyphosphate, coated with hyaluronic acid, and in complex with polycaprolactone. The hydrodynamic size of the nanoparticles by dynamic light scattering, zeta potential, Fourier transform infrared spectroscopy, scanning electron microscopy, stability, loading efficiency, and release kinetics with ovalbumin were evaluated. Balb/c mice were immunized intranasally using the three-dose protocol with nanoparticles, gB, and adjuvants Poly(I:C) and CpG ODN. Subsequently, the humoral and cell-mediated antigen-specific immune response was determined. On the basis of the properties of the tested nanoparticles, the cross-linked nanoparticles were considered optimal for further investigation. The results show that nanoparticles with Poly(I:C) and with gB alone raised IgG antibody levels above the negative control. In the case of mucosal IgA, only gB alone weakly induced the production of IgA antibodies compared to saline-immunized mice. The number of activated cells increased slightly in mice immunized with nanoparticles and gB compared to those immunized with gB alone or to negative control. The results demonstrated that chitosan nanoparticles could have potential in the development of mucosal vaccines.
Assuntos
Quitosana , Muromegalovirus , Nanopartículas , Vacinas , Animais , Camundongos , Quitosana/química , Administração Intranasal , Imunidade nas Mucosas , Imunização , Adjuvantes Imunológicos , Imunoglobulina A , Glicoproteínas , Nanopartículas/química , Camundongos Endogâmicos BALB CRESUMO
Cytomegalovirus (CMV) induces a unique T cell response, where antigen-specific populations do not contract, but rather inflate during viral latency. It has been proposed that subclinical episodes of virus reactivation feed the inflation of CMV-specific memory cells by intermittently engaging T cell receptors (TCRs), but evidence of TCR engagement has remained lacking. Nuclear factor of activated T cells (NFAT) is a family of transcription factors, where NFATc1 and NFATc2 signal downstream of TCR in mature T lymphocytes. We show selective impacts of NFATc1 and/or NFATc2 genetic ablations on the long-term inflation of MCMV-specific CD8+ T cell responses despite largely maintained responses to acute infection. NFATc1 ablation elicited robust phenotypes in isolation, but the strongest effects were observed when both NFAT genes were missing. CMV control was impaired only when both NFATs were deleted in CD8+ T cells used in adoptive immunotherapy of immunodeficient mice. Transcriptome analyses revealed that T cell intrinsic NFAT is not necessary for CD8+ T cell priming, but rather for their maturation towards effector-memory and in particular the effector cells, which dominate the pool of inflationary cells.
Assuntos
Infecções por Citomegalovirus , Muromegalovirus , Animais , Camundongos , Muromegalovirus/fisiologia , Linfócitos T CD8-Positivos , Citomegalovirus , Receptores de Antígenos de Linfócitos T , Memória ImunológicaRESUMO
Conventional antiviral memory CD4 T cells typically arise during the first two weeks of acute infection. Unlike most viruses, cytomegalovirus (CMV) exhibits an extended persistent replication phase followed by lifelong latency accompanied with some gene expression. We show that during mouse CMV (MCMV) infection, CD4 T cells recognizing an epitope derived from the viral M09 protein only develop after conventional memory T cells have already peaked and contracted. Ablating these CD4 T cells by mutating the M09 genomic epitope in the MCMV Smith strain, or inducing them by introducing the epitope into the K181 strain, resulted in delayed or enhanced control of viral persistence, respectively. These cells were shown to be unique compared to their conventional memory counterparts; producing higher IFNγ and IL-2 and lower IL-10 levels. RNAseq analyses revealed them to express distinct subsets of effector genes as compared to classical CD4 T cells. Additionally, when M09 cells were induced by epitope vaccination they significantly enhanced protection when compared to conventional CD4 T cells alone. These data show that late-rising CD4 T cells are a unique memory subset with excellent protective capacities that display a development program strongly differing from the majority of memory T cells.
Assuntos
Infecções por Citomegalovirus , Muromegalovirus , Animais , Camundongos , Linfócitos T CD4-Positivos , Epitopos , Glândulas Salivares , Linfócitos T CD8-PositivosRESUMO
The MHC class I molecule H-2Dk conveys resistance to acute murine CMV infection in both C57L (H-2Dk transgenic) and MA/My mice. M.H2k/b mice are on an MA/My background aside from a C57L-derived region spanning the MHC (Cmv5s), which diminishes this resistance and causes significant spleen histopathology. To hone in on the effector elements within the Cmv5s interval, we generated several Cmv5-recombinant congenic mouse strains and screened them in vivo, allowing us to narrow the phenotype-associated interval >6-fold and segment the genetic mechanism to at least two independent loci within the MHC region. In addition, we sought to further characterize the Cmv5s-associated phenotypes in their temporal appearance and potential direct relationship to viral load. To this end, we found that Cmv5s histopathology and NK cell activation could not be fully mirrored in the MA/My mice with increased viral dose, and that marginal zone destruction was the first apparent Cmv5s phenotype, being reliably quantified as early as 2 d postinfection in the M.H2k/b mice, prior to divergence in viral load, weight loss, or NK cell phenotype. Finally, we further dissect NK cell involvement, finding no intrinsic differences in NK cell function, despite increased upregulation of activation markers and checkpoint receptors. In conclusion, these data dissect the genetic and immunologic underpinnings of Cmv5 and reveal a model in which polymorphism within the MHC region of the genome leads to the development of tissue damage and corrupts protective NK cell immunity during acute viral infection.
Assuntos
Infecções por Citomegalovirus , Muromegalovirus , Camundongos , Animais , Antígenos de Histocompatibilidade Classe I/genética , Células Matadoras Naturais , Tecido Linfoide , Camundongos Endogâmicos C57BLRESUMO
Host control of mouse cytomegalovirus (MCMV) infection of MHCII- salivary gland acinar cells is mediated by CD4+ T cells, but how they protect is unclear. Here, we show CD4+ T cells control MCMV indirectly in the salivary gland, via IFNγ engagement with uninfected, but antigen+ MHCII+ APC and recruitment of NK cells to infected cell foci. This immune mechanism renders direct contact of CD4+ T cells with infected cells unnecessary and may represent a host strategy to overcome viral immune evasion.
Assuntos
Infecções por Citomegalovirus , Muromegalovirus , Camundongos , Animais , Linfócitos T , Citoproteção , Células Matadoras Naturais , Linfócitos T CD4-Positivos , Camundongos Endogâmicos C57BLRESUMO
OBJECTIVE: Compare hearing outcomes utilizing standard, prolonged and delayed ganciclovir (GCV) therapy in a murine model of cytomegalovirus (CMV). METHODS: BALB/c mice were inoculated with mouse cytomegalovirus (mCMV) or saline via intracerebral injection on postnatal day 3 (p3). Intraperitoneal GCV or saline was administered at 12 h intervals for the duration of the standard (p3-p17), delayed (p30-p44), or prolonged treatment windows (p3-p31). Auditory thresholds were assessed using distortion product otoacoustic emission (DPOAE) and auditory brainstem response (ABR) testing at 4, 6, and 8 weeks of age. Blood and tissue samples were harvested from mice on p17 and p37 one hour after GCV administration, and their concentrations were assessed via liquid chromatography-mass spectrometry. RESULTS: A delayed course of GCV improved ABR but not DPOAE thresholds in mCMV-infected mice. A prolonged course of GCV did not provide better hearing thresholds than those administered standard treatment. The average GCV concentration in all 17-day-old mice tissue was significantly higher than those in older 37-day-old mice. CONCLUSION: Delayed GCV treatment provided a hearing benefit on ABR over untreated mCMV infected mice. Prolonged CGV administration showed no benefit compared to a shorter duration GCV treatment. GCV drug concentrations both systemically and in the cochlea are much lower in older mice. These results have potential implications for the clinical management of cCMV infected children. LEVEL OF EVIDENCE: NA Laryngoscope, 134:433-438, 2024.
Assuntos
Infecções por Citomegalovirus , Muromegalovirus , Humanos , Criança , Animais , Camundongos , Idoso , Ganciclovir/uso terapêutico , Infecções por Citomegalovirus/tratamento farmacológico , Citomegalovirus , Emissões Otoacústicas Espontâneas , Camundongos Endogâmicos BALB C , Antivirais/uso terapêuticoRESUMO
Like all herpesviruses, cytomegaloviruses (CMVs) code for many immunomodulatory proteins including chemokines. The human cytomegalovirus (HCMV) CC chemokine pUL128 has a dual role in the infection cycle. On one hand, it forms the pentameric receptor-binding complex gHgLpUL(128,130,131A), which is crucial for the broad cell tropism of HCMV. On the other hand, it is an active chemokine that attracts leukocytes and shapes their activation. All animal CMVs studied so far have functionally homologous CC chemokines. In murine cytomegalovirus (MCMV), the CC chemokine is encoded by the m131/m129 reading frames. The MCMV CC chemokine is called MCK2 and forms a trimeric gHgLMCK2 entry complex. Here, we have generated MCK2 mutant viruses either unable to form gHgLMCK2 complexes, lacking the chemokine function or lacking both functions. By using these viruses, we could demonstrate that gHgLMCK2-dependent entry and MCK2 chemokine activity are independent functions of MCK2 in vitro and in vivo. The gHgLMCK2 complex promotes the tropism for leukocytes like macrophages and dendritic cells and secures high titers in salivary glands in MCMV-infected mice independent of the chemokine activity of MCK2. In contrast, reduced early antiviral T cell responses in MCMV-infected mice are dependent on MCK2 being an active chemokine and do not require the formation of gHgLMCK2 complexes. High levels of CCL2 and IFN-γ in spleens of infected mice and MCMV virulence depend on both, the formation of gHgLMCK2 complexes and the MCK2 chemokine activity. Thus, independent and concerted functions of MCK2 serving as chemokine and part of a gHgL entry complex shape antiviral immunity and virus dissemination.