Murine Cytomegalovirus Research Articles

Virological, innate, and adaptive immune profiles shaped by variation in route and age of host in murine cytomegalovirus infection.

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.

Abstract LB117: Virus-inspired Particles (ViPs) harnessing cytomegalovirus immune memory for local and systemic cancer immunotherapy

Abstract VerImmune is pioneering a First-in-Class treatment known as “Anti-tumor Immune Redirection” (AIR). This approach repurposes a patient’s pre-existing anti-viral or childhood vaccine immunity towards tumor cells for targeted destruction. VerImmune’s lead product, VERI-101, leverages pre-existing CD8+ T-cell immune memory acquired from past human cytomegalovirus (HCMV) infections. VERI-101 consists of VerImmune’s proprietary platform technology known as ViPs (Virus-inspired Particles) that are conjugated on their surface with a CD8+ T cell viral peptide antigen. ViPs are based on a modified mouse papillomavirus capsid protein of which 60 copies self-assemble into a 20-30nm T=1 icosahedral structure. In the case of VERI-101, the ViP is conjugated with an HLA-A*0201 restricted peptide epitope (NLVPMVATV, [NLV]), derived from the CMV pp65 antigen with an upstream furin protease cleavage site. The mechanism of action of AIR-ViPs including VERI-101 has been previously presented and involves three steps. Briefly, (1) VERI-101 targets the surface of the tumor cells and (2) enables the presentation of the CMV viral epitopes on their surface MHC, together (3) stimulating a recall of the pre-existing CMV memory response to attack the cancer cells (Dorrier et al., 2023 AACR). Here, we sought to study the in vivo anti-tumor efficacy of AIR-ViPs via both intra-tumoral and systemic treatment routes in two murine tumor models with pre-existing murine CMV (MCMV) immunity: (1) MC38 subcutaneous tumors for local and (2) B16.F10 lung metastasis for systemic treatments. As human CMV is species-specific, instead of VERI-101, a surrogate product, VERI-003 which redirects MCMV immunity, was tested as a single agent or in combination with anti-PD-1 in these tumor models. Our intra-tumoral studies with VERI-003 alone showed a dose-dependent anti-tumor effect (from 5 to 100ug) compared to no treatment controls. Surprisingly, no dose dependence was observed across the same three dose levels when combined with anti-PD-1, although significantly better anti-tumor effects occurred leading to complete tumor clearances in 50% of mice. Statistically significant prolonged survival was observed in most treated groups compared to controls. Our systemic studies with VERI-003 were consistent with our intratumoral studies in showing statistically significant single agent anti-tumor efficacy against B16.F10 lung metastases compared to no treatment. Anti-tumor effects of anti-PD-1 and combination were also observed. Additional studies are on-going to assess dose dependence and analysis of PK/PD effects. Taken together, our initial in vivo results demonstrate the tumor antigen-agnostic therapeutic potential of AIR and our AIR-ViP technology as a novel class of immuno-therapeutic drugs that could be used either as a monotherapy or in combination with checkpoint inhibitors. Citation Format: Cayce Dorrier, Felagot Abebe, Olivia Pak, Emily De-Bodene, Joshua Wang. Virus-inspired Particles (ViPs) harnessing cytomegalovirus immune memory for local and systemic cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB117.

Modulation of cytomegalovirus immune evasion identifies direct antigen presentation as the predominant mode of CD8 T-cell priming during immune reconstitution after hematopoietic cell transplantation.

Cytomegalovirus (CMV) infection is the most critical infectious complication in recipients of hematopoietic cell transplantation (HCT) in the period between a therapeutic hematoablative treatment and the hematopoietic reconstitution of the immune system. Clinical investigation as well as the mouse model of experimental HCT have consistently shown that timely reconstitution of antiviral CD8 T cells is critical for preventing CMV disease in HCT recipients. Reconstitution of cells of the T-cell lineage generates naïve CD8 T cells with random specificities among which CMV-specific cells need to be primed by presentation of viral antigen for antigen-specific clonal expansion and generation of protective antiviral effector CD8 T cells. For CD8 T-cell priming two pathways are discussed: "direct antigen presentation" by infected professional antigen-presenting cells (pAPCs) and "antigen cross-presentation" by uninfected pAPCs that take up antigenic material derived from infected tissue cells. Current view in CMV immunology favors the cross-priming hypothesis with the argument that viral immune evasion proteins, known to interfere with the MHC class-I pathway of direct antigen presentation by infected cells, would inhibit the CD8 T-cell response. While the mode of antigen presentation in the mouse model of CMV infection has been studied in the immunocompetent host under genetic or experimental conditions excluding either pathway of antigen presentation, we are not aware of any study addressing the medically relevant question of how newly generated naïve CD8 T cells become primed in the phase of lympho-hematopoietic reconstitution after HCT. Here we used the well-established mouse model of experimental HCT and infection with murine CMV (mCMV) and pursued the recently described approach of up- or down-modulating direct antigen presentation by using recombinant viruses lacking or overexpressing the central immune evasion protein m152 of mCMV, respectively. Our data reveal that the magnitude of the CD8 T-cell response directly reflects the level of direct antigen presentation.

Pan-viral propagation blockade by inhibiting host cell PNPT1

Successful viral propagation within infected cells necessitates the viruses' ability to overcome the cellular integrated stress response (ISR), triggered during viral infection, which in turn inhibits general protein translation. In our study, we unveil a shared tactic employed by viruses to suppress 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 eIF2α phosphorylation, resulting in the suppression of translation. Furthermore, by scrutinizing the PNPT1 recognition element and screening 17,728 FDA-approved drugs and bioactive compounds, we identify lanatoside C as a potent PNPT1 inhibitor. This compound robustly impedes the propagation of adenovirus, murine cytomegalovirus, and hepatovirus, along with suppressing the production of the SARS-CoV-2 spike protein. Our discoveries shed light on a novel strategy to impede pan-viral propagation by activating the host cell mt-dsRNA-PKR-eIF2α signaling axis.

Type 1 immunity enables neonatal thymic ILC1 production.

Acute thymic atrophy occurs following type 1 inflammatory conditions such as viral infection and sepsis, resulting in cell death and disruption of T cell development. However, the impact type 1 immunity has on thymic-resident innate lymphoid cells (ILCs) remains unclear. Single-cell RNA sequencing revealed neonatal thymic-resident type 1 ILCs (ILC1s) as a unique and immature subset compared to ILC1s in other primary lymphoid organs. Culturing murine neonatal thymic lobes with the type 1 cytokines interleukin-12 (IL-12) and IL-18 resulted in a rapid expansion and thymic egress of KLRG1+CXCR6+ cytotoxic ILC1s. Live imaging showed the subcapsular thymic localization and exit of ILC1s following IL-12 + IL-18 stimulation. Similarly, murine cytomegalovirus infection in neonates resulted in thymic atrophy and subcapsular localization of thymic-resident ILC1s. Neonatal thymic grafting revealed that type 1 inflammation enhances the homing of cytokine-producing thymus-derived ILC1s to the liver and peritoneal cavity. Together, we show that type 1 immunity promotes the expansion and peripheral homing of thymic-derived ILC1s.

Multiple Immune and Genetic Mechanisms Contribute to Cmv5s-Driven Susceptibility and Tissue Damage during Acute Murine Cytomegalovirus Infection.

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 invivo, 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.

Cell-type dependence of necroptosis pathways triggered by viral infection.

Necroptosis, a potent host defense mechanism, limits viral replication and pathogenesis through three distinct initiation pathways. Toll-like receptor 3 (TLR3) via TIR-domain-containing adapter-inducing interferon-β (TRIF), Z-DNA-binding protein 1 (ZBP1) and tumor necrosis factor (TNF)α mediate necroptosis, with ZBP1 and TNF playing pivotal roles in controlling viral infections, with the role of TLR3-TRIF being less clear. ZBP1-mediated necroptosis is initiated when host ZBP1 senses viral Z-form double stranded RNA and recruits receptor-interacting serine/threonine-protein kinase 3 (RIPK3), driving a mixed lineage kinase domain-like pseudokinase (MLKL)-dependent necroptosis pathway, whereas TNF-mediated necroptosis is initiated by TNF signaling, which drives a RIPK1-RIPK3-MLKL pathway, resulting in necroptosis. Certain viruses (cytomegalovirus, herpes simplex virus and vaccinia) have evolved to produce proteins that compete with host defense systems, preventing programmed cell death pathways from being initiated. Two engineered viruses deficient of active forms of these proteins, murine cytomegalovirus M45mutRHIM and vaccinia virus E3∆Zα, trigger ZBP1-dependent necroptosis in mouse embryonic fibroblasts. By contrast, when bone-marrow-derived macrophages are infected with the viruses, necroptosis is initiated predominantly through the TNF-mediated pathway. However, when the TNF pathway is blocked by RIPK1 inhibitors or a TNF blockade, ZBP1-mediated necroptosis becomes the prominent pathway in bone-marrow-derived macrophages. Overall, these data implicate a cell-type preference for either TNF-mediated or ZBP1-mediated necroptosis pathways in host responses to viral infections. These preferences are important to consider when evaluating disease models that incorporate necroptosis because they may contribute to tissue-specific reactions that could alter the balance of inflammation versus control of virus, impacting the organism as a whole.

Caspase cleavage of RIPK3 after Asp333 is dispensable for mouse embryogenesis

The proteolytic activity of caspase-8 suppresses lethal RIPK1-, RIPK3- and MLKL-dependent necroptosis during mouse embryogenesis. Caspase-8 is reported to cleave RIPK3 in addition to the RIPK3-interacting kinase RIPK1, but whether cleavage of RIPK3 is crucial for necroptosis suppression is unclear. Here we show that caspase-8-driven cleavage of endogenous mouse RIPK3 after Asp333 is dependent on downstream caspase-3. Consistent with RIPK3 cleavage being a consequence of apoptosis rather than a critical brake on necroptosis, Ripk3D333A/D333A knock-in mice lacking the Asp333 cleavage site are viable and develop normally. Moreover, in contrast to mice lacking caspase-8 in their intestinal epithelial cells, Ripk3D333A/D333A mice do not exhibit increased sensitivity to high dose tumor necrosis factor (TNF). Ripk3D333A/D333A macrophages died at the same rate as wild-type (WT) macrophages in response to TNF plus cycloheximide, TNF plus emricasan, or infection with murine cytomegalovirus (MCMV) lacking M36 and M45 to inhibit caspase-8 and RIPK3 activation, respectively. We conclude that caspase cleavage of RIPK3 is dispensable for mouse development, and that cleavage of caspase-8 substrates, including RIPK1, is sufficient to prevent necroptosis.

Intermittent cytomegalovirus infection alters neurobiological metabolism and induces cognitive deficits in mice

Risk factors contributing to dementia are multifactorial. Accumulating evidence suggests a role for pathogens as risk factors, but data is largely correlative with few causal relationships. Here, we demonstrate that intermittent murine cytomegalovirus (MCMV) infection of mice, alters blood brain barrier (BBB) permeability and metabolic pathways. Increased basal mitochondrial function is observed in brain microvessels cells (BMV) exposed to intermittent MCMV infection and is accompanied by elevated levels of superoxide. Further, mice score lower in cognitive assays compared to age-matched controls who were never administered MCMV. Our data show that repeated systemic infection with MCMV, increases markers of neuroinflammation, alters mitochondrial function, increases markers of oxidative stress and impacts cognition. Together, this suggests that viral burden may be a risk factor for dementia. These observations provide possible mechanistic insights through which pathogens may contribute to the progression or exacerbation of dementia.

A single-dose MCMV-based vaccine elicits long-lasting immune protection in mice against distinct SARS-CoV-2 variants.

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.

Targeting the tissue factor coagulation initiation complex prevents antiphospholipid antibody development

AbstractAntiphospholipid antibodies (aPL) in primary or secondary antiphospholipid syndrome (APS) are a major cause for acquired thrombophilia, but specific interventions preventing autoimmune aPL development are an unmet clinical need. Although autoimmune aPL cross react with various coagulation regulatory proteins, lipid-reactive aPL, including those derived from patients with COVID-19, recognize the endolysosomal phospholipid lysobisphosphatidic acid presented by the cell surface–expressed endothelial protein C receptor. This specific recognition leads to complement-mediated activation of tissue factor (TF)–dependent proinflammatory signaling and thrombosis. Here, we show that specific inhibition of the TF coagulation initiation complex with nematode anticoagulant protein c2 (NAPc2) prevents the prothrombotic effects of aPL derived from patients with COVID-19 in mice and the aPL–induced proinflammatory and prothrombotic activation of monocytes. The induction of experimental APS is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, and NAPc2 suppresses monocyte endosomal reactive oxygen species production requiring the TF cytoplasmic domain and interferon-α secretion from dendritic cells. Latent infection with murine cytomegalovirus causes TF cytoplasmic domain–dependent development of persistent aPL and circulating phospholipid-reactive B1 cells, which is prevented by short-term intervention with NAPc2 during acute viral infection. In addition, treatment of lupus prone MRL-lpr mice with NAPc2, but not with heparin, suppresses dendritic-cell activation in the spleen, aPL production and circulating phospholipid-reactive B1 cells, and attenuates lupus pathology. These data demonstrate a convergent TF-dependent mechanism of aPL development in latent viral infection and autoimmune disease and provide initial evidence that specific targeting of the TF initiation complex has therapeutic benefits beyond currently used clinical anticoagulant strategies.

A systematic analysis of anthocyanins inhibiting human, murine, and equine herpesviruses

BackgroundHerpesviruses are common animal and human pathogens that cause severe health problems in children, immunocompromised patients, and infected animals with a host range from fish to mammals. Anthocyanin-containing plant extracts have been described as potent antivirals, which might cause fewer harmful side effects than direct-acting antivirals. Here, we report that an extract of Aristotelia chilensis (Molina) Stuntz (Elaeocarpaceae) (MBE) with a high content of the anthocyanin delphinidin suppresses lytic replication of equine, murine and human herpesviruses of replication in vitro. MethodsWe treated cultured cells with MBE and purified individual anthocyanins present in the extract to determine the most active compound at different concentrations. We subsequently infected the cultures with human herpesviruses 1 (HSV-1) or 8 (HHV-8), murine cytomegalovirus (CMV), or equine herpesviruses 1 (EHV-1) and determined the number of infected cells and viral infectivity. ResultsMBE inhibited the HSV-1, murine CMV, and EHV-1 by up to 2 orders of magnitude. In the presence of the stabilizing randomly methylated-beta-cyclodextrin, the inhibitory concentration could be lowered significantly. We identified delphinidin as an active antiviral compound and showed that the non-glycosylated delphinidin solved and stabilized with sulfobutylether-beta-cyclodextrin allowed usage of approximately 50 times lower concentrations. ConclusionGlycosylated delphinidin derivatives were identified as active antiviral compounds of MBE. This suggests that plant extracts rich in delphinidin-anthocyanins have potent antiviral properties that could be used in treatment and prevention

Nasal Immunization Using Chitosan Nanoparticles with Glycoprotein B of Murine Cytomegalovirus.

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.

Direct antigen presentation is the canonical pathway of cytomegalovirus CD8 T-cell priming regulated by balanced immune evasion ensuring a strong antiviral response.

CD8 T cells are important antiviral effectors in the adaptive immune response to cytomegaloviruses (CMV). Naïve CD8 T cells can be primed by professional antigen-presenting cells (pAPCs) alternatively by "direct antigen presentation" or "antigen cross-presentation". In the case of direct antigen presentation, viral proteins are expressed in infected pAPCs and enter the classical MHC class-I (MHC-I) pathway of antigen processing and presentation of antigenic peptides. In the alternative pathway of antigen cross-presentation, viral antigenic material derived from infected cells of principally any cell type is taken up by uninfected pAPCs and eventually also fed into the MHC class-I pathway. A fundamental difference, which can be used to distinguish between these two mechanisms, is the fact that viral immune evasion proteins that interfere with the cell surface trafficking of peptide-loaded MHC-I (pMHC-I) complexes are absent in cross-presenting uninfected pAPCs. Murine cytomegalovirus (mCMV) models designed to disrupt either of the two presentation pathways revealed that both are possible in principle and can substitute each other. Overall, however, the majority of evidence has led to current opinion favoring cross-presentation as the canonical pathway. To study priming in the normal host genetically competent in both antigen presentation pathways, we took the novel approach of enhancing or inhibiting direct antigen presentation by using recombinant viruses lacking or overexpressing a key mCMV immune evasion protein. Against any prediction, the strongest CD8 T-cell response was elicited under the condition of intermediate direct antigen presentation, as it exists for wild-type virus, whereas the extremes of enhanced or inhibited direct antigen presentation resulted in an identical and weaker response. Our findings are explained by direct antigen presentation combined with a negative feedback regulation exerted by the newly primed antiviral effector CD8 T cells. This insight sheds a completely new light on the acquisition of viral immune evasion genes during virus-host co-evolution.

Dissecting the cytomegalovirus CC chemokine: Chemokine activity and gHgLchemokine-dependent cell tropism are independent players in CMV infection.

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.

Circulating Exosomal miRNA Signature As a Potential Risk Factor for DNA Virus Infection in Patients with Multiple Myeloma

Introduction DNA virus infection is a significant cause of morbidity and mortality in patients with multiple myeloma (MM). Monocyte dysfunction in MM patients plays a central role in infectious complications, but the precise molecular mechanism underlying the reduced monocyte resistance to viruses and risk factors for infection in newly diagnosed MM patients remains to be elucidated. Methods To determine the role of exosomal miRNAs in DNA infection of MM patients, we analyzed miRNAs in BM exosomes from MM patients (Exo-miRs) using high-throughput small RNA sequencing. To further evaluate the effect of these five upregulated Exo-miRs in antiviral response, we transfected the host cells with chemically synthesized miRNAs mimics to overexpress miRNAs. We utilized qPCR and WB to characterize the fate the cGAS-STING signaling pathway of THP-1 after overexpress miRNAs. The efficacy and safety of these Exo-miRs in innate antiviral immunity were evaluated using 5TGM1 C57BL/KaLwRij mice models. Additionally, miRNAs copy number as well as the viral susceptibility of newly diagnosed MM patients were determined in 82 samples from newly diagnosed biopsy proven MM patients. Results Screening of miRNAs enriched in exosomes delivered by primary MM cells identified five miRNAs as inhibitory factors that negatively regulate the cGAS-STING antiviral immune response. When engraftments of 5TGM1-Luc cells via tail vein for eight weeks in C57BL/KaLwRij mice resulted in MM progression with typical MM phenotypes, model mice were primed with Exo-miR antagomiRs oligonucleotides mixture through tail vein injection three times every 24 h followed by Murine cytomegalovirus (MCMV) challenge for 36 h. Consistent with the immunosuppression in MM patients, 5TGM1 C57BL/KaLwRij mice showed lower IFN-β in sera. Notably, intravenous administration of antagomiRs against miRNAs resulted in a marked increase of IFN-β levels in serum and IFN-I expression in organs (Figure 1). To investigate the potential of the miR-score as a prognostic determinant of vulnerability to DNA virus infection, we compared the miR-score in plasma exosomes collected from 82 newly diagnosed biopsy proven MM patients. We classified the viral susceptibility of newly diagnosed MM patients into two groups: I, DNA virus infection; II, no DNA virus infection. In human, a pronounced inverse correlation between IFN-β production by DNA virus in PBMCs and the microRNA (miR)-score of the exosomes within the plasma. The patients with DNA virus infection showed significantly enhanced circulatory expression of miRNAs than without DNA virus infection (p<0.001) patients. The diagnostic potential of miRNAs was assessed by plotting ROC curve, 5 miRNAs showed optimum sensitivity and specificity（AUC 0.854, P<0.0001, Sensitivity 87.5%, Specificity 74.1%, Cut-off values 5.234)（Figure 2）. Conclusions These findings identify a mechanism by which MM cells can suppress the host's innate immunity and potentially lead to immunocompromise in MM patients. Furthermore, the aberrant expression of specific miRNAs in MM patients emerges as a potential therapeutic target in MM cells. By quantifying miRNA scores based on abundant exosomal miRNAs in peripheral blood, the risk of DNA virus infection in MM patients can be assessed. Our findings shed light on a previously unknown molecular mechanism underlying immunosuppression in MM patients who have not received antineoplastic therapy, and also reveal a distinct miRNA signature that serves as a crucial determinant of the susceptibility of newly diagnosed MM patients to DNA virus infections.

Comparison Between Two Types of Viral-Induced Anterior Uveitis In Vitro and In Vivo: A Stronger Response in Herpes Simplex Virus Type 1 Than in Murine Cytomegalovirus.

To observe the similarities and differences between herpes simplex virus type 1 (HSV-1) and murine cytomegalovirus (MCMV)-induced viral anterior uveitis (VAU), both in vitro and in vivo. Primary rat trabecular meshwork cells (RTMCs) were infected by HSV-1 or MCMV to clarify the pattern of virus replication and the effect on cells. In vivo, intracameral injection of HSV-1 or MCMV was performed to establish the VAU rat models. The clinical manifestation, intraocular pressure (IOP), histological characteristics, ultrastructural changes, and the expression of inflammatory cytokines in the anterior segment were observed and compared between these two types of VAU models. Both viruses could infect the RTMCs but HSV-1 exhibited an earlier and greater cytopathic effect in vitro. In vivo, both VAU rats showed typical acute VAU signs, and the IOP elevation seemed to be correlated with the inflammatory progression. Histopathological findings and ultrastructural changes revealed tissue damage and cell infiltration in the anterior chamber angle. In both models, similar proinflammatory cytokines were upregulated. HSV-1 and MCMV viral particles were identified under transmission electron microscopy. HSV-1 and MCMV infection share certain similarities but have significant differences both in vitro and in vivo. HSV-1 usually has a stronger anterior segment inflammation with a longer duration compared with MCMV in VAU models. Our results provided a valuable animal model for investigating pathogenesis and exploring therapeutic strategies for clinical VAU.

Immunotherapy of cytomegalovirus infection by low-dose adoptive transfer of antiviral CD8 T cells relies on substantial post-transfer expansion of central memory cells but not effector-memory cells.

Cytomegaloviruses (CMVs) are host species-specific in their replication. It is a hallmark of all CMVs that productive primary infection is controlled by concerted innate and adaptive immune responses in the immunocompetent host. As a result, the infection usually passes without overt clinical symptoms and develops into latent infection, referred to as "latency". During latency, the virus is maintained in a non-replicative state from which it can reactivate to productive infection under conditions of waning immune surveillance. In contrast, infection of an immunocompromised host causes CMV disease with viral multiple-organ histopathology resulting in organ failure. Primary or reactivated CMV infection of hematopoietic cell transplantation (HCT) recipients in a "window of risk" between therapeutic hemato-ablative leukemia therapy and immune system reconstitution remains a clinical challenge. Studies in the mouse model of experimental HCT and infection with murine CMV (mCMV), followed by clinical trials in HCT patients with human CMV (hCMV) reactivation, have revealed a protective function of virus-specific CD8 T cells upon adoptive cell transfer (AT). Memory CD8 T cells derived from latently infected hosts are a favored source for immunotherapy by AT. Strikingly low numbers of these cells were found to prevent CMV disease, suggesting either an immediate effector function of few transferred cells or a clonal expansion generating high numbers of effector cells. In the murine model, the memory population consists of resting central memory T cells (TCM), as well as of conventional effector-memory T cells (cTEM) and inflationary effector-memory T cells (iTEM). iTEM increase in numbers over time in the latently infected host, a phenomenon known as 'memory inflation' (MI). They thus appeared to be a promising source for use in immunotherapy. However, we show here that iTEM contribute little to the control of infection after AT, which relies almost entirely on superior proliferative potential of TCM.

Viral infection causes long term changes of the immunological milieu within the heart

Abstract Background The last two decades have brought plentiful insights and new advances in understanding virus-host interactions, and the importance of acute and latent viral infections in the modulation of the structure and function of the mammalian immune system became apparent. Despite abundant evidence demonstrating substential changes after viral infections in the immune cell composition and the local immune response within various organs such as the lungs and the gut, the same phenomena within the heart tissue has remained poorly investigated. Methods C57BL6/J 6–12-week-old female mice were infected intravenously with murine cytomegalovirus (MCMV) or left uninfected. Kinetics of viral replication within the heart were determined using a standard plaque assay. Furthermore, immune cell composition within the heart was determined by flow cytometry during acute and latent viral infection. Results Active MCMV replication within the heart peaked at day 5 post-infection (p.i.) and the replicating viral particles became undetectable by day 12 in all of the infected animals. The initial immune cell infiltration was led by the increase in the NK cell compartment, followed by a substantial increase in the T cell compartment which peaked around day 7 p.i. Furthermore, there were substantial phenotypic changes in the population of cardiac macrophages which became predominantly MHCIIhigh and Ly6Chigh. Most notable was an enlargement within the CD8 T cell population which increased around 60-fold compared to the steady state conditions. Crucially, the changes in the immune cell milieu were maintained long after cessation of active viral replication as the hearts of latently infected mice contained around 2.5 times more leukocytes (CD45+) than the hearts of non-infected animals, around 18% percent of which were CD8 T cells, compared to 5% observed in the mock-infected group. Conclusion Systemic viral infection with a ubiquitous herpesvirus causes long term alterations in the composition and phenotype of the heart immune cells which could have major consequences for myocardial coping with different pathological injuries such as a subsequent microbial infection, ischemic conditions or stress induced by hypertension or volume overload.

Infection of liver sinusoidal endothelial cells with Muromegalovirus muridbeta1 involves binding to neuropilin-1 and is dynamin-dependent.

Liver sinusoidal endothelial cells (LSEC) are scavenger cells with a remarkably high capacity for clearance of several blood-borne macromolecules and nanoparticles, including some viruses. Endocytosis in LSEC is mainly via the clathrin-coated pit mediated route, which is dynamin-dependent. LSEC can also be a site of infection and latency of betaherpesvirus, but mode of virus entry into these cells has not yet been described. In this study we have investigated the role of dynamin in the early stage of muromegalovirus muridbeta1 (MuHV-1, murid betaherpesvirus 1, murine cytomegalovirus) infection in mouse LSECs. LSEC cultures were freshly prepared from C57Bl/6JRj mouse liver. We first examined dose- and time-dependent effects of two dynamin-inhibitors, dynasore and MitMAB, on cell viability, morphology, and endocytosis of model ligands via different LSEC scavenger receptors to establish a protocol for dynamin-inhibition studies in these primary cells. LSECs were challenged with MuHV-1 (MOI 0.2) ± dynamin inhibitors for 1h, then without inhibitors and virus for 11h, and nuclear expression of MuHV-1 immediate early antigen (IE1) measured by immune fluorescence. MuHV-1 efficiently infected LSECs in vitro. Infection was significantly and independently inhibited by dynasore and MitMAB, which block dynamin function via different mechanisms, suggesting that initial steps of MuHV-1 infection is dynamin-dependent in LSECs. Infection was also reduced in the presence of monensin which inhibits acidification of endosomes. Furthermore, competitive binding studies with a neuropilin-1 antibody blocked LSEC infection. This suggests that MuHV-1 infection in mouse LSECs involves virus binding to neuropilin-1 and occurs via endocytosis.