• A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients binds to the ACE2-RBD interface and is tolerant to most known RBD mutations.

      Bertoglio, Federico; Fühner, Viola; Ruschig, Maximilian; Heine, Philip Alexander; Abassi, Leila; Klünemann, Thomas; Rand, Ulfert; Meier, Doris; Langreder, Nora; Steinke, Stephan; et al. (Cell Press, 2021-07-07)
      The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with FcγR-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19.
    • Targeted T cell receptor gene editing provides predictable T cell product function for immunotherapy.

      Müller, Thomas R; Jarosch, Sebastian; Hammel, Monika; Leube, Justin; Grassmann, Simon; Bernard, Bettina; Effenberger, Manuel; Andrä, Immanuel; Chaudhry, M Zeeshan; Käuferle, Theresa; et al. (Elsevier, 2021-08-17)
      Adoptive transfer of T cells expressing a transgenic T cell receptor (TCR) has the potential to revolutionize immunotherapy of infectious diseases and cancer. However, the generation of defined TCR-transgenic T cell medicinal products with predictable in vivo function still poses a major challenge and limits broader and more successful application of this "living drug." Here, by studying 51 different TCRs, we show that conventional genetic engineering by viral transduction leads to variable TCR expression and functionality as a result of variable transgene copy numbers and untargeted transgene integration. In contrast, CRISPR/Cas9-mediated TCR replacement enables defined, targeted TCR transgene insertion into the TCR gene locus. Thereby, T cell products display more homogeneous TCR expression similar to physiological T cells. Importantly, increased T cell product homogeneity after targeted TCR gene editing correlates with predictable in vivo T cell responses, which represents a crucial aspect for clinical application in adoptive T cell immunotherapy.
    • SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface.

      Bertoglio, Federico; Meier, Doris; Langreder, Nora; Steinke, Stephan; Rand, Ulfert; Simonelli, Luca; Heine, Philip Alexander; Ballmann, Rico; Schneider, Kai-Thomas; Roth, Kristian Daniel Ralph; et al. (NPG, 2021-03-11)
      COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a new recently emerged sarbecovirus. This virus uses the human ACE2 enzyme as receptor for cell entry, recognizing it with the receptor binding domain (RBD) of the S1 subunit of the viral spike protein. We present the use of phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve antibody gene libraries HAL9/10 and subsequent identification of 309 unique fully human antibodies against S1. 17 antibodies are binding to the RBD, showing inhibition of spike binding to cells expressing ACE2 as scFv-Fc and neutralize active SARS-CoV-2 virus infection of VeroE6 cells. The antibody STE73-2E9 is showing neutralization of active SARS-CoV-2 as IgG and is binding to the ACE2-RBD interface. Thus, universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovering patients in a pandemic situation.
    • A Novel Triple-Fluorescent HCMV Strain Reveals Gene Expression Dynamics and Anti-Herpesviral Drug Mechanisms.

      Rand, Ulfert; Kubsch, Tobias; Kasmapour, Bahram; Cicin-Sain, Luka; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2021-01-08)
      Human Cytomegalovirus (HCMV) infection may result in severe outcomes in immunocompromised individuals such as AIDS patients, transplant recipients, and neonates. To date, no vaccines are available and there are only few drugs for anti-HCMV therapy. Adverse effects and the continuous emergence of drug-resistance strains require the identification of new drug candidates in the near future. Identification and characterization of such compounds and biological factors requires sensitive and reliable detection techniques of HCMV infection, gene expression and spread. In this work, we present and validate a novel concept for multi-reporter herpesviruses, identified through iterative testing of minimally invasive mutations. We integrated up to three fluorescence reporter genes into replication-competent HCMV strains, generating reporter HCMVs that allow the visualization of replication cycle stages of HCMV, namely the immediate early (IE), early (E), and late (L) phase. Fluorescent proteins with clearly distinguishable emission spectra were linked by 2A peptides to essential viral genes, allowing bicistronic expression of the viral and the fluorescent protein without major effects on viral fitness. By using this triple color reporter HCMV, we monitored gene expression dynamics of the IE, E, and L genes by measuring the fluorescent signal of the viral gene-associated fluorophores within infected cell populations and at high temporal resolution. We demonstrate distinct inhibitory profiles of foscarnet, fomivirsen, phosphonoacetic acid, ganciclovir, and letermovir reflecting their mode-of-action. In conclusion, our data argues that this experimental approach allows the identification and characterization of new drug candidates in a single step.
    • Triple RNA-Seq Reveals Synergy in a Human Virus-Fungus Co-infection Model.

      Seelbinder, Bastian; Wallstabe, Julia; Marischen, Lothar; Weiss, Esther; Wurster, Sebastian; Page, Lukas; Löffler, Claudia; Bussemer, Lydia; Schmitt, Anna-Lena; Wolf, Thomas; et al. (Elsevier (Cell Press), 2020-11-17)
      High-throughput RNA sequencing (RNA-seq) is routinely applied to study diverse biological processes; however, when performed separately on interacting organisms, systemic noise intrinsic to RNA extraction, library preparation, and sequencing hampers the identification of cross-species interaction nodes. Here, we develop triple RNA-seq to simultaneously detect transcriptomes of monocyte-derived dendritic cells (moDCs) infected with the frequently co-occurring pulmonary pathogens Aspergillus fumigatus and human cytomegalovirus (CMV). Comparing expression patterns after co-infection with those after single infections, our data reveal synergistic effects and mutual interferences between host responses to the two pathogens. For example, CMV attenuates the fungus-mediated activation of pro-inflammatory cytokines through NF-κB (nuclear factor κB) and NFAT (nuclear factor of activated T cells) cascades, while A. fumigatus impairs viral clearance by counteracting viral nucleic acid-induced activation of type I interferon signaling. Together, the analytical power of triple RNA-seq proposes molecular hubs in the differential moDC response to fungal/viral single infection or co-infection that contribute to our understanding of the etiology and, potentially, clearance of post-transplant infections.
    • Synthetic rewiring and boosting type I interferon responses for visualization and counteracting viral infections.

      Gödecke, Natascha; Riedel, Jan; Herrmann, Sabrina; Behme, Sara; Rand, Ulfert; Kubsch, Tobias; Cicin-Sain, Luka; Hauser, Hansjörg; Köster, Mario; Wirth, Dagmar; et al. (Oxford Academic, 2020-11-18)
      Mammalian first line of defense against viruses is accomplished by the interferon (IFN) system. Viruses have evolved numerous mechanisms to reduce the IFN action allowing them to invade the host and/or to establish latency. We generated an IFN responsive intracellular hub by integrating the synthetic transactivator tTA into the chromosomal Mx2 locus for IFN-based activation of tTA dependent expression modules. The additional implementation of a synthetic amplifier module with positive feedback even allowed for monitoring and reacting to infections of viruses that can antagonize the IFN system. Low and transient IFN amounts are sufficient to trigger these amplifier cells. This gives rise to higher and sustained-but optionally de-activatable-expression even when the initial stimulus has faded out. Amplification of the IFN response induced by IFN suppressing viruses is sufficient to protect cells from infection. Together, this interfaced sensor/actuator system provides a toolbox for robust sensing and counteracting viral infections.
    • The avid competitors of memory inflation.

      Abassi, Leila; Cicin-Sain, Luka; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2020-10-08)
      Cytomegaloviruses (CMV) coevolve with their hosts and latently persist in the vast majority of adult mammals. Therefore, persistent T-cell responses to CMV antigens during virus latency offer a fascinating perspective on the evolution of the T-cell repertoire in natural settings. We addressed here the life-long interactions between CMV antigens presented on MHC-I molecules and the CD8 T-cell response. We present the mechanistic evidence from the murine model of CMV infection and put it in context of clinical laboratory results. We will highlight the remarkable parallels in T-cell responses between the two biological systems, and focus in particular on memory inflation as a result of competitive processes, both between viral antigenic peptides and between T-cell receptors on the host’s cytotoxic lymphocytes
    • Cytomegalovirus inhibition of extrinsic apoptosis determines fitness and resistance to cytotoxic CD8 T cells.

      Chaudhry, M Zeeshan; Casalegno-Garduno, Rosaely; Sitnik, Katarzyna M; Kasmapour, Bahram; Pulm, Ann-Kathrin; Brizic, Ilija; Eiz-Vesper, Britta; Moosmann, Andreas; Jonjic, Stipan; Mocarski, Edward S; et al. (National Academy of Sciences, 2020-05-22)
      Viral immune evasion is currently understood to focus on deflecting CD8 T cell recognition of infected cells by disrupting antigen presentation pathways. We evaluated viral interference with the ultimate step in cytotoxic T cell function, the death of infected cells. The viral inhibitor of caspase-8 activation (vICA) conserved in human cytomegalovirus (HCMV) and murine CMV (MCMV) prevents the activation of caspase-8 and proapoptotic signaling. We demonstrate the key role of vICA from either virus, in deflecting antigen-specific CD8 T cell-killing of infected cells. vICA-deficient mutants, lacking either UL36 or M36, exhibit greater susceptibility to CD8 T cell control than mutants lacking the set of immunoevasins known to disrupt antigen presentation via MHC class I. This difference is evident during infection in the natural mouse host infected with MCMV, in settings where virus-specific CD8 T cells are adoptively transferred. Finally, we identify the molecular mechanism through which vICA acts, demonstrating the central contribution of caspase-8 signaling at a point of convergence of death receptor-induced apoptosis and perforin/granzyme-dependent cytotoxicity.
    • 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.
    • Exhaustion and Inflation at Antipodes of T Cell Responses to Chronic Virus Infection.

      Cicin-Sain, Luka; Arens, Ramon; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2017-12-14)
      Viruses that have coevolved with their host establish chronic infections that are well tolerated by the host. Other viruses, that are partly adapted to their host, may induce chronic infections where persistent replication and viral antigen expression occur. The former induce highly functional and resilient CD8T cell responses called memory inflation. The latter induce dysfunctional and exhausted responses. The reasons compelling T cell responses towards inflationary or exhausted responses are only partly understood. In this review we compare the two conditions and describe mechanistic similarities and differences. We also provide a list of potential reasons why exhaustion or inflation occur in different virus infections. We propose that T cell-mediated transcriptional repression of viral gene expression provides a critical feature of inflation that allows peaceful virus and host coexistence. The virus is controlled, but its genome is not eradicated. If this mechanism is not available, as in the case of RNA viruses, the virus and the host are compelled to an arms race. If virus proliferation and spread proceed uncontrolled for too long, T cells are forced to strike a balance between viral control and tissue destruction, losing antiviral potency and facilitating virus persistence.
    • Advances in cytomegalovirus (CMV) biology and its relationship to health, diseases, and aging.

      Nikolich-Žugich, Janko; Čicin-Šain, Luka; Collins-McMillen, Donna; Jackson, Sarah; Oxenius, Annette; Sinclair, John; Snyder, Christopher; Wills, Mark; Lemmermann, Niels (Springer, 2020-03-11)
      The complexity of host-associated microbial ecosystems requires host-specific reference catalogs to survey the functions and diversity of these communities. We generate a comprehensive resource, the integrated mouse gut metagenome catalog (iMGMC), comprising 4.6 million unique genes and 660 metagenome-assembled genomes (MAGs), many (485 MAGs, 73%) of which are linked to reconstructed full-length 16S rRNA gene sequences. iMGMC enables unprecedented coverage and taxonomic resolution of the mouse gut microbiota; i.e., more than 92% of MAGs lack species-level representatives in public repositories (<95% ANI match). The integration of MAGs and 16S rRNA gene data allows more accurate prediction of functional profiles of communities than predictions based on 16S rRNA amplicons alone. Accompanying iMGMC, we provide a set of MAGs representing 1,296 gut bacteria obtained through complementary assembly strategies. We envision that integrated resources such as iMGMC, together with MAG collections, will enhance the resolution of numerous existing and future sequencing-based studies.
    • 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.
    • 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.
    • Life-long control of cytomegalovirus (CMV) by T resident memory cells in the adipose tissue results in inflammation and hyperglycemia.

      Contreras, Nico A; Sitnik, Katarzyna M; Jeftic, Ilija; Coplen, Christopher Patrick; Čičin-Šain, Luka; Nikolich-Žugich, Janko; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (PLOS, 2019-06-01)
      Cytomegalovirus (CMV) is a ubiquitous herpesvirus infecting most of the world's population. CMV has been rigorously investigated for its impact on lifelong immunity and potential complications arising from lifelong infection. A rigorous adaptive immune response mounts during progression of CMV infection from acute to latent states. CD8 T cells, in large part, drive this response and have very clearly been demonstrated to take up residence in the salivary gland and lungs of infected mice during latency. However, the role of tissue resident CD8 T cells as an ongoing defense mechanism against CMV has not been studied in other anatomical locations. Therefore, we sought to identify additional locations of anti-CMV T cell residency and the physiological consequences of such a response. Through RT-qPCR we found that mouse CMV (mCMV) infected the visceral adipose tissue and that this resulted in an expansion of leukocytes in situ. We further found, through flow cytometry, that adipose tissue became enriched in cytotoxic CD8 T cells that are specific for mCMV antigens from day 7 post infection through the lifespan of an infected animal (> 450 days post infection) and that carry markers of tissue residence. Furthermore, we found that inflammatory cytokines are elevated alongside the expansion of CD8 T cells. Finally, we show a correlation between the inflammatory state of adipose tissue in response to mCMV infection and the development of hyperglycemia in mice. Overall, this study identifies adipose tissue as a location of viral infection leading to a sustained and lifelong adaptive immune response mediated by CD8 T cells that correlates with hyperglycemia. These data potentially provide a mechanistic link between metabolic syndrome and chronic infection.
    • Early primed KLRG1- CMV-specific T cells determine the size of the inflationary T cell pool.

      Baumann, Nicolas S; Welten, Suzanne P M; Torti, Nicole; Pallmer, Katharina; Borsa, Mariana; Barnstorf, Isabel; Oduro, Jennifer D; Cicin-Sain, Luka; Oxenius, Annette; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (PLOS, 2019-05-01)
      Memory T cell inflation is a process in which a subset of cytomegalovirus (CMV) specific CD8 T cells continuously expands mainly during latent infection and establishes a large and stable population of effector memory cells in peripheral tissues. Here we set out to identify in vivo parameters that promote and limit CD8 T cell inflation in the context of MCMV infection. We found that the inflationary T cell pool comprised mainly high avidity CD8 T cells, outcompeting lower avidity CD8 T cells. Furthermore, the size of the inflationary T cell pool was not restricted by the availability of specific tissue niches, but it was directly related to the number of virus-specific CD8 T cells that were activated during priming. In particular, the amount of early-primed KLRG1- cells and the number of inflationary cells with a central memory phenotype were a critical determinant for the overall magnitude of the inflationary T cell pool. Inflationary memory CD8 T cells provided protection from a Vaccinia virus challenge and this protection directly correlated with the size of the inflationary memory T cell pool in peripheral tissues. These results highlight the remarkable protective potential of inflationary CD8 T cells that can be harnessed for CMV-based T cell vaccine approaches.
    • Circulating levels of the anti-oxidant indoleproprionic acid are associated with higher gut microbiome diversity.

      Menni, Cristina; Hernandez, Marisa Matey; Vital, Marius; Mohney, Robert P; Spector, Tim D; Valdes, Ana M; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Taylor & Francis, 2019-04-29)
      The gut microbiome has recently emerged as an important regulator of insulin resistance and abdominal obesity. The tryptophan metabolite generated by the gut microbiome, indoleproprionic acid (IPA) has been shown to predict the onset of type 2 diabetes. IPA is a metabolite produced by gut microbes from dietary tryptophan that exhibits a high degree of inter-individual variation. The microbiome composition parameters that are associated with circulating levels of this potent anti-oxidant have however not been investigated to date in human populations. In 1018 middle-aged women from the TwinsUK cohort, we assessed the relationship between serum IPA levels and gut microbiome composition targeting the 16S rRNA gene. Microbiome alpha-diversity was positively correlated with serum indoleproprionic acid levels (Shannon Diversity: Beta[95%CI] = 0.19[0.13;0.25], P = 6.41 × 10-10) after adjustment for covariates. Sixteen taxa and 12 operational taxonomic units (OTUs) associated with IPA serum levels. Among these are positive correlations with the butyrate-producing Faecalibacterium prausnitzii, the class Mollicutes and the order RF39 of the Tenericutes, and Coprococcus Negative correlations instead were observed with Eubacterium dolichum previously shown to correlate with visceral fat mass and several genera in the Lachnospiraceae family such as Blautia and Ruminococcus previously shown to correlate with obesity. Microbiome composition parameters explained ~20% of the variation in circulating levels of IPA, whereas nutritional and host genetic parameters explained only ~4%. Our data confirm an association between IPA circulating levels and metabolic syndrome parameters and indicate that gut microbiome composition influences IPA levels.
    • Demarcated thresholds of tumor-specific CD8 T cells elicited by MCMV-based vaccine vectors provide robust correlates of protection.

      Beyranvand Nejad, Elham; Ratts, Robert B; Panagioti, Eleni; Meyer, Christine; Oduro, Jennifer D; Cicin-Sain, Luka; Früh, Klaus; van der Burg, Sjoerd H; Arens, Ramon; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (BMC, 2019-01-31)
      The capacity of cytomegalovirus (CMV) to elicit long-lasting strong T cell responses, and the ability to engineer the genome of this DNA virus positions CMV-based vaccine vectors highly suitable as a cancer vaccine platform. Defined immune thresholds for tumor protection and the factors affecting such thresholds have not well been investigated in cancer immunotherapy. We here determined using CMV as a vaccine platform whether critical thresholds of vaccine-specific T cell responses can be established that relate to tumor protection, and which factors control such thresholds. We generated CMV-based vaccine vectors expressing the E7 epitope and tested these in preclinical models of HPV16-induced cancer. Vaccination was applied via different doses and routes (intraperitoneal (IP), subcutaneous (SC) and intranasal (IN)). The magnitude, kinetics and phenotype of the circulating tumor-specific CD8 Immunization with CMV-based vaccines via the IP or SC route eliciting vaccine-induced CD8 This study highlight the effectiveness of CMV-based vaccine vectors, and shows that demarcated thresholds of vaccine-specific T cells could be defined that correlate to tumor protection. Together, these results may hold importance for cancer vaccine development to achieve high efficacy in vaccine recipients.
    • Human monocyte-derived macrophages inhibit HCMV spread independent of classical antiviral cytokines.

      Becker, Jennifer; Kinast, Volker; Döring, Marius; Lipps, Christoph; Duran, Veronica; Spanier, Julia; Tegtmeyer, Pia-Katharina; Wirth, Dagmar; Cicin-Sain, Luka; Alcamí, Antonio; et al. (2018-01-01)
      Infection of healthy individuals with human cytomegalovirus (HCMV) is usually unnoticed and results in life-long latency, whereas HCMV reactivation as well as infection of newborns or immunocompromised patients can cause life-threatening disease. To better understand HCMV pathogenesis we studied mechanisms that restrict HCMV spread. We discovered that HCMV-infected cells can directly trigger plasmacytoid dendritic cells (pDC) to mount antiviral type I interferon (IFN-I) responses, even in the absence of cell-free virus. In contrast, monocyte-derived cells only expressed IFN-I when stimulated by cell-free HCMV, or upon encounter of HCMV-infected cells that already produced cell-free virus. Nevertheless, also in the absence of cell-free virus, i.e., upon co-culture of infected epithelial/endothelial cells and monocyte-derived macrophages (moMΦ) or dendritic cells (moDC), antiviral responses were induced that limited HCMV spread. The induction of this antiviral effect was dependent on cell-cell contact, whereas cell-free supernatants from co-culture experiments also inhibited virus spread, implying that soluble factors were critically needed. Interestingly, the antiviral effect was independent of IFN-γ, TNF-α, and IFN-I as indicated by cytokine inhibition experiments using neutralizing antibodies or the vaccinia virus-derived soluble IFN-I binding protein B18R, which traps human IFN-α and IFN-β. In conclusion, our results indicate that human macrophages and dendritic cells can limit HCMV spread by IFN-I dependent as well as independent mechanisms, whereas the latter ones might be particularly relevant for the restriction of HCMV transmission via cell-to-cell spread.
    • The chromatin remodeling factor SPOC1 acts as a cellular restriction factor against human cytomegalovirus by repressing the major immediate-early promoter.

      Reichel, Anna; Stilp, Anne-Charlotte; Scherer, Myriam; Reuter, Nina; Lukassen, Sören; Kasmapour, Bahram; Schreiner, Sabrina; Cicin-Sain, Luka; Winterpacht, Andreas; Stamminger, Thomas; et al. (2018-05-09)
      The cellular protein SPOC1 (survival time-associated PHD finger protein in ovarian cancer 1) acts as a regulator of chromatin structure and DNA damage response. It binds H3K4me2/3 containing chromatin and promotes DNA condensation by recruiting corepressors such as KAP-1 and H3K9 methyltransferases. Previous studies identified SPOC1 as a restriction factor against human adenovirus (HAdV) infection that is antagonized by E1B-55K/E4orf6-dependent proteasomal degradation. Here, we demonstrate that, in contrast to HAdV-infected cells, SPOC1 is transiently upregulated during the early phase of HCMV replication. We show that expression of the immediate-early protein 1 (IE1) is sufficient and necessary to induce SPOC1. Additionally, we discovered that during later stages of infection SPOC1 is downregulated in a GSK-3β-dependent manner. We provide evidence that SPOC1 overexpression severely impairs HCMV replication by repressing the initiation of viral immediate early (IE) gene expression. Consistently, we observed that SPOC1-depleted primary human fibroblasts displayed augmented initiation of viral IE gene expression. This occurs in a MOI-dependent manner, a defining hallmark of intrinsic immunity. Interestingly, repression requires the presence of high SPOC1 levels at the start of infection while a later upregulation had no negative impact suggesting distinct temporal roles of SPOC1 during the HCMV replicative cycle. Mechanistically, we observed a highly specific association of SPOC1 with the major immediate-early promoter (MIEP) strongly suggesting that SPOC1 inhibits HCMV replication by MIEP binding and subsequent recruitment of heterochromatin building factors. Thus, our data add SPOC1 as a novel factor to the endowment of a host cell to restrict cytomegalovirus infections.
    • 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.