• Mouse CMV infection delays antibody class switch upon an unrelated virus challenge.

      Marandu, Thomas F; Finsterbusch, Katja; Kröger, Andrea; Čičin-Šain, Luka; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2014-06)
      Poor immune protection upon vaccination is a critical determinant of immunosenescence. Latent Cytomegalovirus (CMV) infection has been associated with poor antibody responses to vaccination, but a causative role for CMV in the poor immune response requires experimental evidence and thus could not be confirmed in clinical studies. To test the hypothesis that latent CMV infection causes poor antibody responses, we infected young or adult mice with mouse CMV and challenged them with Vesicular stomatitis virus (VSV) at 15 or 18months of age. Latent, but not primary infection with mouse CMV resulted in diminished neutralizing titers of the serum IgG fraction at day 7 post challenge, which recovered by day 14 post challenge. This phenomenon was specific for mice infected with mouse CMV, but not mice infected with other herpesviruses, like murine herpesvirus-68 or herpes simplex virus type 1, or mice infected with non-persistent viruses, such as influenza or Vaccinia virus. Hence, our data indicate a delay in IgG class-switch that was specific for the CMV infection. Herpesviral infections did not change the B-cell memory compartment, and increased the size of the effector-memory subset of blood CD4 T-cells only when administered in combination. Furthermore, CD4 T-cell response to VSV infection was maintained in latently infected mice. Therefore, our results argue that latent CMV infection impairs B-cell, but not T-cell responses to a challenge with VSV and delays antibody class-switch by a mechanism which may be independent of T-cell help.
    • Mucosal CD8+ T cell responses induced by an MCMV based vaccine vector confer protection against influenza challenge.

      Zheng, Xiaoyan; Oduro, Jennifer D; Boehme, Julia D; Borkner, Lisa; Ebensen, Thomas; Heise, Ulrike; Gereke, Marcus; Pils, Marina C; Krmpotic, Astrid; Guzmán, Carlos A; et al. (PLOS, 2019-09-01)
      Cytomegalovirus (CMV) is a ubiquitous β-herpesvirus that establishes life-long latent infection in a high percentage of the population worldwide. CMV induces the strongest and most durable CD8+ T cell response known in human clinical medicine. Due to its unique properties, the virus represents a promising candidate vaccine vector for the induction of persistent cellular immunity. To take advantage of this, we constructed a recombinant murine CMV (MCMV) expressing an MHC-I restricted epitope from influenza A virus (IAV) H1N1 within the immediate early 2 (ie2) gene. Only mice that were immunized intranasally (i.n.) were capable of controlling IAV infection, despite the greater potency of the intraperitoneally (i.p.) vaccination in inducing a systemic IAV-specific CD8+ T cell response. The protective capacity of the i.n. immunization was associated with its ability to induce IAV-specific tissue-resident memory CD8+ T (CD8TRM) cells in the lungs. Our data demonstrate that the protective effect exerted by the i.n. immunization was critically mediated by antigen-specific CD8+ T cells. CD8TRM cells promoted the induction of IFNγ and chemokines that facilitate the recruitment of antigen-specific CD8+ T cells to the lungs. Overall, our results showed that locally applied MCMV vectors could induce mucosal immunity at sites of entry, providing superior immune protection against respiratory infections.
    • Murine cytomegalovirus infection via the intranasal route offers a robust model of immunity upon mucosal CMV infection.

      Oduro, Jennifer D; Redeker, Anke; Lemmermann, Niels A W; Ebermann, Linda; Marandu, Thomas F; Dekhtiarenko, Iryna; Holzki, Julia K; Busch, Dirk; Arens, Ramon; Cicin-Sain, Luka; et al. (2015-11-10)
      Cytomegalovirus (CMV) is a ubiquitous virus, causing the most common congenital infection in humans, yet a vaccine against this virus is not available. The experimental study of immunity against CMV in animal models of infection, such as the infection of mice with the mouse CMV (MCMV), has relied on systemic intraperitoneal infection protocols, although the infection naturally transmits by mucosal routes via body fluids containing CMV. To characterize the biology of infections by mucosal routes, we have compared the kinetics of virus replication, the latent viral load, and CD8 T cell responses in lymphoid organs upon experimental intranasal and intragastric infection to intraperitoneal infection of two unrelated mouse strains. We have observed that intranasal infection induces robust and persistent virus replication in lungs and salivary glands, but a poor one in the spleen. CD8 T cell responses were somewhat weaker than upon intraperitoneal infection, but showed similar kinetic profiles and phenotypes of antigen-specific cells. On the other hand, intragastric infection resulted in abortive or poor virus replication in all tested organs, and poor T cell responses to the virus, especially at late times after infection. Consistent with the T cell kinetics, the MCMV latent load was high in the lungs, but low in the spleen of intranasally infected mice and lowest in all tested organs upon intragastric infection. In conclusion, we show here that intranasal, but not intragastric infection of mice with MCMV represents a robust model to study short and long-term biology of CMV infection by a mucosal route.
    • Myeloid dendritic cells repress human cytomegalovirus gene expression and spread by releasing interferon-unrelated soluble antiviral factors.

      Kasmapour, Bahram; Kubsch, Tobias; Rand, Ulfert; Eiz-Vesper, Britta; Messerle, Martin; Vondran, Florian W R; Wiegmann, Bettina; Haverich, Axel; Cicin-Sain, Luka; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-10-18)
      Cytomegalovirus (CMV) is a beta-herpesvirus that latently infects most adult humans worldwide and is a major cause of morbidity and mortality in immunocompromised hosts. Latent human CMV (HCMV) is believed to reside in precursors of myeloid-lineage, leukocytes and monocytes, which give raise to macrophages and dendritic cells. We report here that human monocyte derived DCs (mo-DC) suppress HCMV infection in coculture with infected fibroblasts target cells in an effector-to-target-ratio dependent manner. Intriguingly, optimal activation of mo-DC was achieved in coculture conditions, not by their direct infection with HCMV, implying that mo-DC may recognize unique molecular patterns on, or within, infected fibroblasts. We show that HCMV is controlled by secreted factors that act by priming defenses in target cells rather than by direct viral neutralization, but we excluded a role for IFNs in this control. The expression of lytic viral genes in infected cells and the progression of infection were significantly slowed down, but this effect was reversible, indicating that the control of infection depended on the transient induction of antiviral effector molecules in target cells. Using immediate-early or late-phase reporter HCMVs, we show that soluble factors secreted in the cocultures suppress HCMV replication at both stages of the infection and that their antiviral effect is robust and comparable in numerous batches of mo-DCs as well as in primary fibroblasts and stromal cells.Importance Human cytomegalovirus is a widespread opportunistic pathogen that can cause severe disease and complications in vulnerable individuals. This includes newborn children, HIV AIDS patients or transplant recipients. Although the majority of healthy humans carry this virus throughout their lives without symptoms, it is not exactly clear which tissues in the body are the main reservoirs of latent virus infection, or how the delicate balance between the virus and the immune system is maintained over the individual's lifetime. Here for the first time, we provide evidence for a novel mechanism of direct virus control by a subset of human innate immune cells called Dendritic Cells, which are regarded as a major site of virus latency and reactivation. Our findings may have important implications in HCMV disease prevention as well as development of novel therapeutic approaches.
    • Peptide Processing Is Critical for T-Cell Memory Inflation and May Be Optimized to Improve Immune Protection by CMV-Based Vaccine Vectors.

      Dekhtiarenko, Iryna; Ratts, Robert B; Blatnik, Renata; Lee, Lian N; Fischer, Sonja; Borkner, Lisa; Oduro, Jennifer D; Marandu, Thomas F; Hoppe, Stephanie; Ruzsics, Zsolt; et al. (2016-12)
      Cytomegalovirus (CMV) elicits long-term T-cell immunity of unparalleled strength, which has allowed the development of highly protective CMV-based vaccine vectors. Counterintuitively, experimental vaccines encoding a single MHC-I restricted epitope offered better immune protection than those expressing entire proteins, including the same epitope. To clarify this conundrum, we generated recombinant murine CMVs (MCMVs) encoding well-characterized MHC-I epitopes at different positions within viral genes and observed strong immune responses and protection against viruses and tumor growth when the epitopes were expressed at the protein C-terminus. We used the M45-encoded conventional epitope HGIRNASFI to dissect this phenomenon at the molecular level. A recombinant MCMV expressing HGIRNASFI on the C-terminus of M45, in contrast to wild-type MCMV, enabled peptide processing by the constitutive proteasome, direct antigen presentation, and an inflation of antigen-specific effector memory cells. Consequently, our results indicate that constitutive proteasome processing of antigenic epitopes in latently infected cells is required for robust inflationary responses. This insight allows utilizing the epitope positioning in the design of CMV-based vectors as a novel strategy for enhancing their efficacy.
    • A replicating cytomegalovirus-based vaccine encoding a single Ebola virus nucleoprotein CTL epitope confers protection against Ebola virus.

      Tsuda, Yoshimi; Caposio, Patrizia; Parkins, Christopher J; Botto, Sara; Messaoudi, Ilhem; Cicin-Sain, Luka; Feldmann, Heinz; Jarvis, Michael A; Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America. (2011-08)
      Human outbreaks of Ebola virus (EBOV) are a serious human health concern in Central Africa. Great apes (gorillas/chimpanzees) are an important source of EBOV transmission to humans due to increased hunting of wildlife including the 'bush-meat' trade. Cytomegalovirus (CMV) is an highly immunogenic virus that has shown recent utility as a vaccine platform. CMV-based vaccines also have the unique potential to re-infect and disseminate through target populations regardless of prior CMV immunity, which may be ideal for achieving high vaccine coverage in inaccessible populations such as great apes.
    • Reversible silencing of cytomegalovirus genomes by type I interferon governs virus latency.

      Dağ, Franziska; Dölken, Lars; Holzki, Julia; Drabig, Anja; Weingärtner, Adrien; Schwerk, Johannes; Lienenklaus, Stefan; Conte, Ianina; Geffers, Robert; Davenport, Colin; et al. (2014-02)
      Herpesviruses establish a lifelong latent infection posing the risk for virus reactivation and disease. In cytomegalovirus infection, expression of the major immediate early (IE) genes is a critical checkpoint, driving the lytic replication cycle upon primary infection or reactivation from latency. While it is known that type I interferon (IFN) limits lytic CMV replication, its role in latency and reactivation has not been explored. In the model of mouse CMV infection, we show here that IFNβ blocks mouse CMV replication at the level of IE transcription in IFN-responding endothelial cells and fibroblasts. The IFN-mediated inhibition of IE genes was entirely reversible, arguing that the IFN-effect may be consistent with viral latency. Importantly, the response to IFNβ is stochastic, and MCMV IE transcription and replication were repressed only in IFN-responsive cells, while the IFN-unresponsive cells remained permissive for lytic MCMV infection. IFN blocked the viral lytic replication cycle by upregulating the nuclear domain 10 (ND10) components, PML, Sp100 and Daxx, and their knockdown by shRNA rescued viral replication in the presence of IFNβ. Finally, IFNβ prevented MCMV reactivation from endothelial cells derived from latently infected mice, validating our results in a biologically relevant setting. Therefore, our data do not only define for the first time the molecular mechanism of IFN-mediated control of CMV infection, but also indicate that the reversible inhibition of the virus lytic cycle by IFNβ is consistent with the establishment of CMV latency.
    • Seropositivity for pathogens associated with chronic infections is a risk factor for all-cause mortality in the elderly: findings from the Memory and Morbidity in Augsburg Elderly (MEMO) Study.

      Zeeb, Marius; Kerrinnes, Tobias; Cicin-Sain, Luka; Guzman, Carlos A; Puppe, Wolfram; Schulz, Thomas F; Peters, Annette; Berger, Klaus; Castell, Stefanie; Karch, André; et al. (Springer, 2020-07-09)
      Immunostimulation by chronic infection has been linked to an increased risk for different non-communicable diseases, which in turn are leading causes of death in high- and middle-income countries. Thus, we investigated if a positive serostatus for pathogens responsible for common chronic infections is individually or synergistically related to reduced overall survival in community dwelling elderly. We used data of 365 individuals from the German MEMO (Memory and Morbidity in Augsburg Elderly) cohort study with a median age of 73 years at baseline and a median follow-up of 14 years. We examined the effect of a positive serostatus at baseline for selected pathogens associated with chronic infections (Helicobacter pylori, Borrelia burgdorferi sensu lato, Toxoplasma gondii, cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1/2, and human herpesvirus 6) on all-cause mortality with multivariable parametric survival models. We found a reduced survival time in individuals with a positive serostatus for Helicobacter pylori (accelerated failure time (AFT) - 15.92, 95% CI - 29.96; - 1.88), cytomegalovirus (AFT - 22.81, 95% CI - 36.41; - 9.22) and Borrelia burgdorferi sensu lato (AFT - 25.25, 95% CI - 43.40; - 7.10), after adjusting for potential confounders. The number of infectious agents an individual was seropositive for had a linear effect on all-cause mortality (AFT per additional infection - 12.42 95% CI - 18.55; - 6.30). Our results suggest an effect of seropositivity for Helicobacter pylori, cytomegalovirus, and Borrelia burgdorferi sensu lato on all-cause mortality in older community dwelling individuals. Further research with larger cohorts and additional biomarkers is required, to assess mediators and molecular pathways of this effect.
    • Tissue maintenance of CMV-specific inflationary memory T cells by IL-15.

      Baumann, Nicolas S; Torti, Nicole; Welten, Suzanne P M; Barnstorf, Isabel; Borsa, Mariana; Pallmer, Katharina; Oduro, Jennifer D; Cicin-Sain, Luka; Ikuta, Koichi; Ludewig, Burkhard; et al. (2018-04)
      Cytomegalovirus (CMV) infection induces an atypical CD8 T cell response, termed inflationary, that is characterised by accumulation and maintenance of high numbers of effector memory like cells in circulation and peripheral tissues-a feature being successfully harnessed for vaccine purposes. Although stability of this population depends on recurrent antigen encounter, the requirements for prolonged survival in peripheral tissues remain unknown. Here, we reveal that murine CMV-specific inflationary CD8 T cells are maintained in an antigen-independent manner and have a half-life of 12 weeks in the lung tissue. This half-life is drastically longer than the one of phenotypically comparable inflationary effector cells. IL-15 alone, and none of other common γ-cytokines, was crucial for survival of inflationary cells in peripheral organs. IL-15, mainly produced by non-hematopoietic cells in lung tissue and being trans-presented, promoted inflationary T cell survival by increasing expression of Bcl-2. These results indicate that inflationary CD8 T cells are not just simply effector-like cells, rather they share properties of both effector and memory CD8 T cells and they appear to be long-lived cells compared to the effector cells from acute virus infections.
    • UL36 Rescues Apoptosis Inhibition and In vivo Replication of a Chimeric MCMV Lacking the M36 Gene.

      Chaudhry, M Zeeshan; Kasmapour, Bahram; Plaza-Sirvent, Carlos; Bajagic, Milica; Casalegno Garduño, Rosaely; Borkner, Lisa; Lenac Roviš, Tihana; Scrima, Andrea; Jonjic, Stipan; Schmitz, Ingo; et al. (2017)
      Apoptosis is an important defense mechanism mounted by the immune system to control virus replication. Hence, cytomegaloviruses (CMV) evolved and acquired numerous anti-apoptotic genes. The product of the human CMV (HCMV) UL36 gene, pUL36 (also known as vICA), binds to pro-caspase-8, thus inhibiting death-receptor apoptosis and enabling viral replication in differentiated THP-1 cells. In vivo studies of the function of HCMV genes are severely limited due to the strict host specificity of cytomegaloviruses, but CMV orthologues that co-evolved with other species allow the experimental study of CMV biology in vivo. The mouse CMV (MCMV) homolog of the UL36 gene is called M36, and its protein product (pM36) is a functional homolog of vICA that binds to murine caspase-8 and inhibits its activation. M36-deficient MCMV is severely growth impaired in macrophages and in vivo. Here we show that pUL36 binds to the murine pro-caspase-8, and that UL36 expression inhibits death-receptor apoptosis in murine cells and can replace M36 to allow MCMV growth in vitro and in vivo. We generated a chimeric MCMV expressing the UL36 ORF sequence instead of the M36 one. The newly generated MCMV(UL36) inhibited apoptosis in macrophage lines RAW 264.7, J774A.1, and IC-21 and its growth was rescued to wild type levels. Similarly, growth was rescued in vivo in the liver and spleen, but only partially in the salivary glands of BALB/c and C57BL/6 mice. In conclusion, we determined that an immune-evasive HCMV gene is conserved enough to functionally replace its MCMV counterpart and thus allow its study in an in vivo setting. As UL36 and M36 proteins engage the same molecular host target, our newly developed model can facilitate studies of anti-viral compounds targeting pUL36 in vivo.
    • Vaccine Vectors Harnessing the Power of Cytomegaloviruses.

      Ynga-Durand, Mario Alberto; Dekhtiarenko, Iryna; Cicin-Sain, Luka; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2019-10-17)
      Cytomegalovirus (CMV) species have been gaining attention as experimental vaccine vectors inducing cellular immune responses of unparalleled strength and protection. This review outline the strengths and the restrictions of CMV-based vectors, in light of the known aspects of CMV infection, pathogenicity and immunity. We discuss aspects to be considered when optimizing CMV based vaccines, including the innate immune response, the adaptive humoral immunity and the T-cell responses. We also discuss the antigenic epitopes presented by unconventional major histocompatibility complex (MHC) molecules in some CMV delivery systems and considerations about routes for delivery for the induction of systemic or mucosal immune responses. With the first clinical trials initiating, CMV-based vaccine vectors are entering a mature phase of development. This impetus needs to be maintained by scientific advances that feed the progress of this technological platform.