Andreas Busche

Recombinant mouse cytomegalovirus expressing a ligand for the NKG2D receptor is attenuated and has improved vaccine properties

Human CMV (HCMV) is a major cause of morbidity and mortality in both congenitally infected and immunocompromised individuals. Development of an effective HCMV vaccine would help protect these vulnerable groups. NK group 2, member D (NKG2D) is a potent activating receptor expressed by cells of the innate and adaptive immune systems. Its importance in HCMV immune surveillance is indicated by the elaborative evasion mechanisms evolved by the virus to avoid NKG2D. In order to study this signaling pathway, we engineered a recombinant mouse CMV expressing the high-affinity NKG2D ligand RAE-1γ (RAE-1γMCMV). Expression of RAE-1γ by MCMV resulted in profound virus attenuation in vivo and lower latent viral DNA loads. RAE-1γMCMV infection was efficiently controlled by immunodeficient hosts, including mice lacking type I interferon receptors or immunosuppressed by sublethal γ-irradiation. Features of MCMV infection in neonates were also diminished. Despite tight innate immune control, RAE-1γMCMV infection elicited strong and long-lasting protective immunity. Maternal RAE-1γMCMV immunization protected neonatal mice from MCMV disease via placental transfer of antiviral Abs. Despite strong selective pressure, the RAE-1γ transgene did not exhibit sequence variation following infection. Together, our results indicate that use of a recombinant virus encoding the ligand for an activating NK cell receptor could be a powerful approach to developing a safe and immunogenic HCMV vaccine.

In Vivo Killing Capacity of Cytotoxic T Cells Is Limited and Involves Dynamic Interactions and T Cell Cooperativity

Summary According to in vitro assays, T cells are thought to kill rapidly and efficiently, but the efficacy and dynamics of cytotoxic T lymphocyte (CTL)-mediated killing of virus-infected cells in vivo remains elusive. We used two-photon microscopy to quantify CTL-mediated killing in mice infected with herpesviruses or poxviruses. On average, one CTL killed 2–16 virus-infected cells per day as determined by real-time imaging and by mathematical modeling. In contrast, upon virus-induced MHC class I downmodulation, CTLs failed to destroy their targets. During killing, CTLs remained migratory and formed motile kinapses rather than static synapses with targets. Viruses encoding the calcium sensor GCaMP6s revealed strong heterogeneity in individual CTL functional capacity. Furthermore, the probability of death of infected cells increased for those contacted by more than two CTLs, indicative of CTL cooperation. Thus, direct visualization of CTLs during killing of virus-infected cells reveals crucial parameters of CD8+ T cell immunity.

Conditional and reversible disruption of essential herpesvirus proteins

Elucidating the function of essential proteins of complex pathogenic viruses is impeded by a paucity of complementing systems. By fusing a destabilizing domain of the FK506-binding protein to essential cytomegalovirus proteins, we generated virus mutants in which amounts of fusion proteins and viral growth can be regulated by the synthetic ligand shield-1. This conditional approach will greatly facilitate the analysis of gene functions of herpesviruses and viruses of other families.

Priming of CD8+ T Cells against Cytomegalovirus Encoded Antigens Is Dominated by Cross Presentation

CMV can infect dendritic cells (DCs), and direct Ag presentation could, therefore, lead to the priming of CMV-specific CD8+ T cells. However, CMV-encoded immune evasins severely impair Ag presentation in the MHC class I pathway; thus, it is widely assumed that cross-presentation drives the priming of antiviral T cells. We assessed the contribution of direct versus cross priming in mouse CMV (MCMV) infection using recombinant viruses. DCs infected with an MCMV strain encoding the gB498 epitope from HSV-1 were unable to stimulate in vitro naive gB498-specific CD8+ T cells from TCR transgenic mice. Infection of C57BL/6 mice with this recombinant virus led, however, to the generation of abundant numbers of gB498-specific T cells in vivo. Of the DC subsets isolated from infected mice, only CD8α+ DCs were able to stimulate naive T cells, suggesting that this DC subset cross-presents MCMV-encoded Ag in vivo. Upon infection of mice with MCMV mutants encoding Ag that can either be well or hardly cross-presented, mainly CD8+ T cells specific for cross-presented epitopes were generated. Moreover, even in the absence of immune evasion genes interfering with MHC class I–mediated Ag presentation, priming of T cells to Ag that can only be presented directly was not observed. We conclude that the host uses mainly DCs capable of cross-presentation to induce the CMV-specific CD8+ T cell response during primary, acute infection and discuss the implications for the development of a CMV vaccine.

Single cell detection of latent cytomegalovirus reactivation in host tissue

The molecular mechanisms leading to reactivation of latent cytomegalovirus are not well understood. To study reactivation, the few cells in an organ tissue that give rise to reactivated virus need to be identified, ideally at the earliest possible time point in the process. To this end, mouse cytomegalovirus (MCMV) reporter mutants were designed to simultaneously express the red fluorescent protein mCherry and the secreted Gaussia luciferase (Gluc). Whereas Gluc can serve to assess infection at the level of individual mice by measuring luminescence in blood samples or by in vivo imaging, mCherry fluorescence offers the advatage of detection of infection at the single cell level. To visualize cells in which MCMV was being reactivated, precision-cut lung slices (PCLS) that preserve tissue microanatomy were prepared from the lungs of latently infected mice. By day 3 of cultivation of the PCLS, reactivation was revealed by Gluc expression, preceding the detection of infectious virus by approximately 4 days. Reactivation events in PCLS could be identified when they were still confined to single cells. Notably, using fractalkine receptor-GFP reporter mice, we never observed reactivation originating from CX3CR1(+) monocytes or pulmonary dendritic cells derived therefrom. Furthermore, latent viral genome in the lungs was not enriched in sorted bone-marrow-derived cells expressing CD11b. Taken together, these complementary approaches suggest that CD11b(+) and CX3CR1(+) subsets of the myeloid differentiation lineage are not the main reservoirs and cellular sites of MCMV latency and reactivation in the lungs.

Nodular inflammatory foci are sites of T cell priming and control of murine cytomegalovirus infection in the neonatal lung.

Neonates, including mice and humans, are highly susceptible to cytomegalovirus (CMV) infection. However, many aspects of neonatal CMV infections such as viral cell tropism, spatio-temporal distribution of the pathogen as well as genesis of antiviral immunity are unknown. With the use of reporter mutants of the murine cytomegalovirus (MCMV) we identified the lung as a primary target of mucosal infection in neonatal mice. Comparative analysis of neonatal and adult mice revealed a delayed control of virus replication in the neonatal lung mucosa explaining the pronounced systemic infection and disease in neonates. This phenomenon was supplemented by a delayed expansion of CD8+ T cell clones recognizing the viral protein M45 in neonates. We detected viral infection at the single-cell level and observed myeloid cells forming “nodular inflammatory foci” (NIF) in the neonatal lung. Co-localization of infected cells within NIFs was associated with their disruption and clearance of the infection. By 2-photon microscopy, we characterized how neonatal antigen-presenting cells (APC) interacted with T cells and induced mature adaptive immune responses within such NIFs. We thus define NIFs of the neonatal lung as niches for prolonged MCMV replication and T cell priming but also as sites of infection control.

Phenotypes of major immediate-early gene mutants of mouse cytomegalovirus

Immediate-early (IE) genes are the first genes to be transcribed during the lytic replication cycle of cytomegaloviruses (CMV), and encode nonstructural proteins, which are assumed to have mainly regulatory functions. The IE proteins may play important roles in the pathogenesis of CMV in vivo, for instance during the establishment of latency and during reactivation. We constructed mouse CMV mutants with disruptions in the major IE genes, ie1 and ie3, to study the roles of these genes in the context of the viral infection. Here we summarize the current results on the characterization of these mutants and give a perspective of the future research in this field.

Reversible Inhibition of Murine Cytomegalovirus Replication by Gamma Interferon (IFN-γ) in Primary Macrophages Involves a Primed Type I IFN-Signaling Subnetwork for Full Establishment of an Immediate-Early Antiviral State

ABSTRACT Activated macrophages play a central role in controlling inflammatory responses to infection and are tightly regulated to rapidly mount responses to infectious challenge. Type I interferon (alpha/beta interferon [IFN-α/β]) and type II interferon (IFN-γ) play a crucial role in activating macrophages and subsequently restricting viral infections. Both types of IFNs signal through related but distinct signaling pathways, inducing a vast number of interferon-stimulated genes that are overlapping but distinguishable. The exact mechanism by which IFNs, particularly IFN-γ, inhibit DNA viruses such as cytomegalovirus (CMV) is still not fully understood. Here, we investigate the antiviral state developed in macrophages upon reversible inhibition of murine CMV by IFN-γ. On the basis of molecular profiling of the reversible inhibition, we identify a significant contribution of a restricted type I IFN subnetwork linked with IFN-γ activation. Genetic knockout of the type I-signaling pathway, in the context of IFN-γ stimulation, revealed an essential requirement for a primed type I-signaling process in developing a full refractory state in macrophages. A minimal transient induction of IFN-β upon macrophage activation with IFN-γ is also detectable. In dose and kinetic viral replication inhibition experiments with IFN-γ, the establishment of an antiviral effect is demonstrated to occur within the first hours of infection. We show that the inhibitory mechanisms at these very early times involve a blockade of the viral major immediate-early promoter activity. Altogether our results show that a primed type I IFN subnetwork contributes to an immediate-early antiviral state induced by type II IFN activation of macrophages, with a potential further amplification loop contributed by transient induction of IFN-β.

Superior induction and maintenance of protective CD8 T cells in mice infected with mouse cytomegalovirus vector expressing RAE-1γ

Significance There are a number of pathogens for which the immunity acquired postinfection does not fully protect against reinfection and disease. Therefore, vaccines offering superior protection compared with the protection following natural infection are needed. Due to a unique pattern of immune response induced by cytomegaloviruses (CMVs), live attenuated CMVs are attractive candidates for vaccine vectors. Here we have demonstrated that a recombinant CMV vector expressing RAE-1γ, a cellular ligand for activating NKG2D receptor expressed on several types of immune cells, has tremendous potential for subverting viral immunoevasion and enhancing the efficiency of the CD8 T-cell response against vectored antigens. This study demonstrates a significant new approach in designing T-cell–based vaccine vectors. Due to a unique pattern of CD8 T-cell response induced by cytomegaloviruses (CMVs), live attenuated CMVs are attractive candidates for vaccine vectors for a number of clinically relevant infections and tumors. NKG2D is one of the most important activating NK cell receptors that plays a role in costimulation of CD8 T cells. Here we demonstrate that the expression of CD8 T-cell epitope of Listeria monocytogenes by a recombinant mouse CMV (MCMV) expressing the NKG2D ligand retinoic acid early-inducible protein 1-gamma (RAE-1γ) dramatically enhanced the effectiveness and longevity of epitope-specific CD8 T-cell response and conferred protection against a subsequent challenge infection with Listeria monocytogenes. Unexpectedly, the attenuated growth in vivo of the CMV vector expressing RAE-1γ and its capacity to enhance specific CD8 T-cell response were preserved even in mice lacking NKG2D, implying additional immune function for RAE-1γ beyond engagement of NKG2D. Thus, vectors expressing RAE-1γ represent a promising approach in the development of CD8 T-cell–based vaccines.

The Mouse Cytomegalovirus Glycoprotein m155 Inhibits CD40 Expression and Restricts CD4 T Cell Responses

ABSTRACT Cytomegaloviruses (CMV) utilize a variety of immunomodulatory strategies to facilitate the establishment of lifelong persistence in their infected hosts. We show that the mouse CMV (MCMV) m155 open reading frame (ORF) is required for the posttranscriptional inhibition of CD40 expression in infected antigen-presenting cells. Consistent with the known importance of CD40-mediated costimulation of T cells, a m155-deficient virus induces enhanced MCMV epitope-specific CD4 T cell responses.