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COVID-19 vaccines prevent severe forms of the disease, but do not warrant complete pro-tection against breakthrough infections. This could be due to suboptimal mucosal immu-nity at the site of virus entry, given that all currently approved vaccines are administeredvia the intramuscular route. In this study, we assessed humoral and cellular immuneresponses in BALB/c mice after intranasal and intramuscular immunization with adenovi-ral vector ChAdOx1-S expressing full- length Spike protein of SARS-CoV-2. We showed thatboth routes of vaccination induced a potent IgG antibody response, as well as robust neu- tralizing capacity, but intranasal vaccination elicited a superior IgA antibody titer in thesera and in the respiratory mucosa. Bronchoalveolar lavage from intranasally immunizedmice efficiently neutralized SARS-CoV-2, which has not been the case in intramuscularlyimmunized group. Moreover, substantially higher percentages of epitope- specific CD8 Tcells exhibiting a tissue resident phenotype were found in the lungs of intranasally immu-nized animals. Finally, both intranasal and intramuscular vaccination with ChAdOx1- Sefficiently protected the mice after the challenge with recombinant herpesvirus express-ing the Spike protein. Our results demonstrate that intranasal application of adenoviralvector ChAdOx1-S induces superior mucosal immunity and therefore could be a promis-ing strategy for putting the COVID-19 pandemic under control.

The twentieth century witnessed a huge expansion in the number of vaccines used with great success in combating diseases, especially the ones caused by viral and bacterial pathogens. Despite this, several major public health threats, such as HIV, tuberculosis, malaria, and cancer, still pose an enormous humanitarian and economic burden. As vaccines based on the induction of protective, neutralizing antibodies have not managed to effectively combat these diseases, in recent decades, the focus has increasingly shifted towards the cellular immune response. There is substantial evidence demonstrating CD8 T cells as key players in the protection not only against many viral and bacterial pathogens, but also in the fight against neoplastic cells. Here, we present arguments for CD8 T cells to be considered as promising candidates for vaccine targeting. We discuss the heterogeneity of CD8 T cell populations and their contribution in the protection of the host. We also outline several strategies of using a common human pathogen, cytomegalovirus, as a vaccine vector since accumulated data strongly suggest it represents a promising approach to the development of novel vaccines against both pathogens and tumors.

Using a mouse model of congenital cytomegalovirus infection, evidence is provided for a pathogenic role of NK and ILC1 cells in the brain. Although brain-infiltrating innate immune cells fail to control infection, they orchestrate pathological inflammatory responses that lead to altered cerebellar development., Congenital human cytomegalovirus (cHCMV) infection of the brain is associated with a wide range of neurocognitive sequelae. Using infection of newborn mice with mouse cytomegalovirus (MCMV) as a reliable model that recapitulates many aspects of cHCMV infection, including disseminated infection, CNS infection, altered neurodevelopment, and sensorineural hearing loss, we have previously shown that mitigation of inflammation prevented alterations in cerebellar development, suggesting that host inflammatory factors are key drivers of neurodevelopmental defects. Here, we show that MCMV infection causes a dramatic increase in the expression of the microglia-derived chemokines CXCL9/CXCL10, which recruit NK and ILC1 cells into the brain in a CXCR3-dependent manner. Surprisingly, brain-infiltrating innate immune cells not only were unable to control virus infection in the brain but also orchestrated pathological inflammatory responses, which lead to delays in cerebellar morphogenesis. Our results identify NK and ILC1 cells as the major mediators of immunopathology in response to virus infection in the developing CNS, which can be prevented by anti–IFN-γ antibodies., Graphical Abstract

Cytomegalovirus (CMV) infection is a significant public health problem. Congenital CMV infection is a leading infectious cause of long-term neurodevelopmental sequelae, including mental retardation and sensorineural hearing loss. Immune protection against mouse cytomegalovirus (MCMV) is primarily mediated by NK cells and CD8+ T cells, while CD4+ T cells are not needed for control of MCMV in majority of organs in immunocompetent adult mice. Here, we set out to determine the role of CD4+ T cells upon MCMV infection of newborn mice. We provide evidence that CD4+ T cells are essential for clearance of MCMV infection in brain of neonatal mice and for prevention of recurrence of latent MCMV. In addition, we provide evidence that CD4+ T cells are required for induction and maintenance of tissue- resident memory CD8+ T cells in the brain of mice perinatally infected with MCMV.

Viral infections during pregnancy are a considerable cause of adverse outcomes and birth defects, and the underlying mechanisms are poorly understood. Among those, cytomegalovirus (CMV) infection stands out as the most common intrauterine infection in humans, putatively causing early pregnancy loss. We employed murine CMV as a model to study the consequences of viral infection on pregnancy outcome and fertility maintenance. Even though pregnant mice successfully controlled CMV infection, we observed highly selective, strong infection of corpus luteum (CL) cells in their ovaries. High infection densities indicated complete failure of immune control in CL cells, resulting in progesterone insufficiency and pregnancy loss. An abundance of gap junctions, absence of vasculature, strong type I interferon (IFN) responses, and interaction of innate immune cells fully protected the ovarian follicles from viral infection. Our work provides fundamental insights into the effect of CMV infection on pregnancy loss and mechanisms protecting fertility.

Viral infections are controlled, and very often cleared, by activated T lymphocytes. The inducible co-stimulator (ICOS) mediates its functions by binding to its ligand ICOSL, enhancing T-cell activation and optimal germinal center (GC) formation. Here, we show that ICOSL is heavily downmodulated during infection of antigen-presenting cells by different herpesviruses. We found that, in murine cytomegalovirus (MCMV), the immunoevasin m138/fcr-1 physically interacts with ICOSL, impeding its maturation and promoting its lysosomal degradation. This viral protein counteracts T-cell responses, in an ICOS-dependent manner, and limits virus control during the acute MCMV infection. Additionally, we report that blockade of ICOSL in MCMV-infected mice critically regulates the production of MCMV-specific antibodies due to a reduction of T follicular helper and GC B cells. Altogether, these findings reveal a novel mechanism evolved by MCMV to counteract adaptive immune surveillance, and demonstrates a role of the ICOS:ICOSL axis in the host defense against herpesviruses., eLife, 10, ISSN:2050-084X

Cytomegaloviruses (CMVs) are ubiquitous pathogens known to employ numerous immunoevasive strategies that significantly impair the ability of the immune system to eliminate the infected cells. Here, we report that the single mouse CMV (MCMV) protein, m154, downregulates multiple surface molecules involved in the activation and costimulation of the immune cells. We demonstrate that m154 uses its cytoplasmic tail motif, DD, to interfere with the adaptor protein-1 (AP-1) complex, implicated in intracellular protein sorting and packaging. As a consequence of the perturbed AP-1 sorting, m154 promotes lysosomal degradation of several proteins involved in T cell costimulation, thus impairing virus-specific CD8+ T cell response and virus control in vivo. Additionally, we show that HCMV infection similarly interferes with the AP-1 complex. Altogether, we identify the robust mechanism employed by single viral immunomodulatory protein targeting a broad spectrum of cell surface molecules involved in the antiviral immune response.

Natural killer (NK) cells are a subset of innate lymphoid cells (ILC) capable of recognizing stressed and infected cells through multiple germ line-encoded receptor-ligand interactions. Missing-self recognition involves NK cell sensing of the loss of host-encoded inhibitory ligands on target cells, including MHC class I (MHC-I) molecules and other MHC-I-independent ligands. Mouse cytomegalovirus (MCMV) infection promotes a rapid host-mediated loss of the inhibitory NKR-P1B ligand Clr-b (encoded by ) on infected cells. Here we provide evidence that an MCMV m145 family member, m153, functions to stabilize cell surface Clr-b during MCMV infection. Ectopic expression of m153 in fibroblasts augments Clr-b cell surface levels. Moreover, infections using -deficient MCMV mutants (Δm144-m158 and Δm153) show an accelerated and exacerbated Clr-b downregulation. Importantly, enhanced loss of Clr-b during Δm153 mutant infection reverts to wild-type levels upon exogenous m153 complementation in fibroblasts. While the effects of m153 on Clr-b levels are independent of transcription, imaging experiments revealed that the m153 and Clr-b proteins only minimally colocalize within the same subcellular compartments, and tagged versions of the proteins were refractory to coimmunoprecipitation under mild-detergent conditions. Surprisingly, the Δm153 mutant possesses enhanced virulence , independent of both Clr-b and NKR-P1B, suggesting that m153 potentially targets additional host factors. Nevertheless, the present data highlight a unique mechanism by which MCMV modulates NK ligand expression. Cytomegaloviruses are betaherpesviruses that in immunocompromised individuals can lead to severe pathologies. These viruses encode various gene products that serve to evade innate immune recognition. NK cells are among the first immune cells that respond to CMV infection and use germ line-encoded NK cell receptors (NKR) to distinguish healthy from virus-infected cells. One such axis that plays a critical role in NK recognition involves the inhibitory NKR-P1B receptor, which engages the host ligand Clr-b, a molecule commonly lost on stressed cells (“missing-self”). In this study, we discovered that mouse CMV utilizes the m153 glycoprotein to circumvent host-mediated Clr-b downregulation, in order to evade NK recognition. These results highlight a novel MCMV-mediated immune evasion strategy.

Železnjak et al. demonstrate that two MCMV-encoded proteins interact with MHC I molecules, forming an altered-self complex that prevents missing self recognition by increasing specificity for inhibitory Ly49 receptors. This led to the evolution of CMV-specific activating Ly49s., CMVs efficiently target MHC I molecules to avoid recognition by cytotoxic T cells. However, the lack of MHC I on the cell surface renders the infected cell susceptible to NK cell killing upon missing self recognition. To counter this, mouse CMV (MCMV) rescues some MHC I molecules to engage inhibitory Ly49 receptors. Here we identify a new viral protein, MATp1, that is essential for MHC I surface rescue. Rescued altered-self MHC I molecules show increased affinity to inhibitory Ly49 receptors, resulting in inhibition of NK cells despite substantially reduced MHC I surface levels. This enables the virus to evade recognition by licensed NK cells. During evolution, this novel viral immune evasion mechanism could have prompted the development of activating NK cell receptors that are specific for MATp1-modified altered-self MHC I molecules. Our study solves a long-standing conundrum of how MCMV avoids recognition by NK cells, unravels a fundamental new viral immune evasion mechanism, and demonstrates how this forced the evolution of virus-specific activating MHC I–restricted Ly49 receptors., Graphical Abstract

Cytomegalovirus (CMV) infection is a significant public health problem. Congenital CMV infection is a leading infectious cause of long-term neurodevelopmental sequelae, including mental retardation and sensorineural hearing loss. Immune protection against mouse cytomegalovirus (MCMV) is primarily mediated by NK cells and CD8(+) T cells, while CD4(+) T cells are not needed for control of MCMV in majority of organs in immunocompetent adult mice. Here we set out to determine the role of CD4(+) T cells upon MCMV infection of newborn mice. We provide evidence that CD4(+) T cells are essential for clearance of MCMV infection in brain of neonatal mice and for prevention of recurrence of latent MCMV. In addition, we provide evidence that CD4(+) T cells are required for induction and maintenance of tissue resident memory CD8(+) T cells in the brain of mice perinatally infected with MCMV.

Cytomegaloviruses (CMVs) are master manipulators of the host immune response. Here, we reveal that the murine CMV (MCMV) protein m152 specifically targets the type I interferon (IFN) response by binding to stimulator of interferon genes (STING), thereby delaying its trafficking to the Golgi compartment from where STING initiates type I IFN signaling. Infection with an MCMV lacking m152 induced elevated type I IFN responses and this leads to reduced viral transcript levels both in vitro and in vivo This effect is ameliorated in the absence of STING Interestingly, while m152 inhibits STING-mediated IRF signaling, it did not affect STING-mediated NF-κB signaling. Analysis of how m152 targets STING translocation reveals that STING activates NF-κB signaling already from the ER prior to its trafficking to the Golgi. Strikingly, this response is important to promote early MCMV replication. Our results show that MCMV has evolved a mechanism to specifically antagonize the STING-mediated antiviral IFN response, while preserving its pro-viral NF-κB response, providing an advantage in the establishment of an infection.

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., Author summary Vaccines against influenza typically induce immune responses based on antibodies, small molecules that recognize the virus particles outside of cells and neutralize them before they infect a cell. However, influenza rapidly evolves, escaping immune recognition, and the fastest evolution is seen in the part of the virus that is recognized by antibodies. Therefore, every year we are confronted with new flu strains that are not recognized by our antibodies against the strains from previous years. The other branch of the immune system is made of killer T cells, which recognize infected cells and target them for killing. Influenza does not rapidly evolve to escape T cell killing; thus, vaccines inducing T-cell responses to influenza might provide long-term protection. We introduced an antigen from influenza into the murine cytomegalovirus (MCMV) and used it as a vaccine vector inducing killer T-cell responses of unparalleled strength. Our vector controls influenza replication and provides relief to infected mice, but only if we administered it through the nose, to activate killer T cells that will persist in the lungs close to the airways. Therefore, our data show that the subset of lung-resident killer T cells is sufficient to protect against influenza.

Cytomegalovirus (CMV) has a high prevalence worldwide, is often fatal for immunocompromised patients, and causes bone marrow suppression. Deficiency of signal transducer and activator of transcription 1 (STAT1) results in severely impaired antiviral immunity. We have used cell- type restricted deletion of Stat1 to determine the importance of myeloid cell activity for the defense against murine CMV (MCMV). We show that myeloid STAT1 limits MCMV burden and infection- associated pathology in the spleen but does not affect ultimate clearance of infection. Unexpectedly, we found an essential role of myeloid STAT1 in the induction of extramedullary hematopoiesis (EMH). The EMH- promoting function of STAT1 was not restricted to MCMV infection but was also observed during CpG oligodeoxynucleotide-induced sterile inflammation. Collectively, we provide genetic evidence that signaling through STAT1 in myeloid cells is required to restrict MCMV at early time points post-infection and to induce compensatory hematopoiesis in the spleen.

Cytomegaloviruses (CMVs) are master manipulators of the host immune response. Here, we reveal that the murine CMV (MCMV) protein m152 specifically targets the type I interferon (IFN) response by binding to stimulator of interferon genes (STING), thereby delaying its trafficking to the Golgi compartment from where STING initiates type I IFN signaling. Infection with an MCMV lacking m152 induced elevated type I IFN responses and this leads to reduced viral transcript levels both in vitro and in vivo. This effect is ameliorated in the absence of STING. Interestingly, while m152 inhibits STING‐mediated IRF signaling, it did not affect STING‐mediated NF‐κB signaling. Analysis of how m152 targets STING translocation reveals that STING activates NF‐κB signaling already from the ER prior to its trafficking to the Golgi. Strikingly, this response is important to promote early MCMV replication. Our results show that MCMV has evolved a mechanism to specifically antagonize the STING‐mediated antiviral IFN response, while preserving its pro‐viral NF‐κB response, providing an advantage in the establishment of an infection.

Congenital HCMV infection is a leading infectious cause of long-term neurodevelopmental sequelae. Infection of newborn mice with MCMV intraperitoneally is a well-established model of congenital HCMV infection, which best recapitulates the hematogenous route of virus spread to brain and subsequent pathology. Here we used this model to investigate the role, dynamics and phenotype of CD8(+) T cells in brain following infection of newborn mice. We show that CD8(+) T cells infiltrate the brain and form a pool of tissue-resident memory T cells (T(RM) cells) that persist for lifetime. Adoptively transferred virus-specific CD8(+) T cells provide protection against primary MCMV infection in newborn mice, reduce brain pathology and remain in the brain as T(RM) cells. Brain CD8(+) T(RM) cells were long-lived, slowly proliferating cells able to respond to local challenge infection. Importantly, brain CD8(+) T(RM) cells controlled latent MCMV and their depletion resulted in virus reactivation and enhanced inflammation in brain.

Congenital HCMV infection is a leading infectious cause of long‐term neurodevelopmental sequelae. Infection of newborn mice with mouse cytomegalovirus (MCMV) intraperitoneally is a well‐established model of congenital human cytomegalovirus infection, which best recapitulates the hematogenous route of virus spread to brain and subsequent pathology. Here, we used this model to investigate the role, dynamics, and phenotype of CD8+ T cells in the brain following infection of newborn mice. We show that CD8+ T cells infiltrate the brain and form a pool of tissue‐resident memory T cells (TRM cells) that persist for lifetime. Adoptively transferred virus‐specific CD8+ T cells provide protection against primary MCMV infection in newborn mice, reduce brain pathology, and remain in the brain as TRM cells. Brain CD8+ TRM cells were long‐ lived, slowly proliferating cells able to respond to local challenge infection. Importantly, brain CD8+ TRM cells controlled latent MCMV and their depletion resulted in virus reactivation and enhanced inflammation in brain.

Cytomegalovirus (CMV) has a high prevalence worldwide, is often fatal for immunocompromised patients and causes bone marrow suppression. Deficiency for signal transducer and activator of transcription 1 (STAT1) results in severely impaired antiviral immunity. We have used cell-type restricted deletion of Stat1 to determine the importance of macrophage activity for the defence against murine CMV (MCMV). We show that macrophage STAT1 limits MCMV burden and infection-associated pathology in the spleen, but does not impact on ultimate clearance of infection. Surprisingly, we found an essential role of macrophage STAT1 in the induction of extramedullary hematopoiesis (EMH). The EMH-promoting function of STAT1 was not only observed after MCMV infection but also during CpG oligodeoxynucleotide-induced sterile inflammation. Collectively, we provide the first genetic evidence that signalling through STAT1 in macrophages is required to restrict MCMV at early time points post infection and to induce compensatory hematopoiesis in the spleen.

Natural killer (NK) and CD8(+) T cells play a crucial role in the control of mouse cytomegalovirus (MCMV) infection. These effector cells exert their functions by releasing antiviral cytokines and by cytolytic mechanisms including perforin activation. In addition to their role in virus control, NK cells play an immunoregulatory role since they shape the CD8(+) T cell response to MCMV. To investigate the role of perforin-dependent cytolytic mechanism in NK cell modulation of CD8(+) T cell response during acute MCMV infection, we have used perforin-deficient C57BL/6 mice (Prf1(-/-)) and have shown that virus control by CD8(+) T cells in Prf1(-/-) mice is more efficient if NK cells are activated by the engagement of the Ly49H receptor with the m157 MCMV protein. A lack of perforin results in severe liver inflammation after MCMV infection, which is characterized by immunopathological lesions that are more pronounced in Prf1(-/-) mice infected with virus unable to activate NK cells. This immunopathology is caused by an abundant infiltration of activated CD8(+) T cells. The depletion of CD8(+) T cells has markedly reduced pathohistological lesions in the liver and improved the survival of Prf1(-/-) mice in spite of an increased viral load. Altogether, the results of our study suggest that a lack of perforin and absence of the specific activation of NK cells during acute MCMV infection lead to an unleashed CD8(+) T cell response that is detrimental for the host.

In contrast to C57BL/6 mice, BALB/c mice are relatively resistant to the induction of experimental autoimmune encephalomyelitis (EAE) after challenge with MOG35–55 peptide. Here, we provide the first evidence that infection with murine cytomegalovirus (MCMV) in adulthood abrogates this resistance. Infected BALB/c mice developed clinical and histological signs similar to those seen in susceptible C57BL/6 mice. In addition to CD4+ cells, large proportion of cells in the infiltrate of diseased BALB/c mice was CD8+, similar with findings in multiple sclerosis. CD8+ cells that responded to ex vivo restimulation with MOG35–55 were not specific for viral epitopes pp89 and m164. MCMV infection favors proinflammatory type of dendritic cells (CD86+CD40+CD11c+) in the peripheral lymph organs, M1 type of microglia in central nervous system, and increases development of Th1/Th17 encephalitogenic cells. This study indicates that MCMV may enhance autoimmune neuropathology and abrogate inherent resistance to EAE in mouse strain by enhancing proinflammatory phenotype of antigen-presenting cells, Th1/Th17, and CD8 response to MOG35–55.

Designing of CD8 T cell vaccines which would provide protection against tumors is still considered a great challenge in immunotherapy. Here we show a robust potential of a cytomegalovirus (CMV) vector expressing the NKG2D ligand RAE-1γ as CD8 T cell-based vaccine against malignant tumors. Immunization with the CMV vector expressing RAE-1γ delayed tumor growth or even provided complete protection against tumor challenge in both prophylactic and therapeutic settings. Moreover, a potent tumor control in mice vaccinated with this vector can be further enhanced by blocking the immune checkpoints TIGIT and PD-1. Expression of RAE-1γ by the CMV vector potentiated expansion of KLRG1+ CD8 T cells with enhanced effector properties. This vaccination was even more efficient in neonatal mice, resulting in the expansion and long-term maintenance of epitope-specific CD8 T cells conferring robust resistance against tumor challenge. Our data show that immunomodulation of CD8 T cell responses promoted by herpesvirus expressing a ligand for NKG2D receptor can provide a powerful platform for the prevention and treatment of CD8 T cell-sensitive tumors.

Natural killer (NK) cells play a key role in innate immunity by detecting alterations in self and non-self ligands via paired NK cell receptors (NKRs). Despite identification of numerous NKR- ligand interactions, physiological ligands for the prototypical NK1.1 orphan receptor remain elusive. Here, we identify a viral ligand for the inhibitory and activating NKRP1(NK1.1) receptors. This murine cytomegalovirus(MCMV)-encoded protein, m12, restrains NK cell effector function by directly engaging the inhibitory NKR-P1B receptor. However, m12 also interacts with the activating NKR-P1A/C receptors to counterbalance m12 decoy function. Structural analyses reveal that m12 sequesters a large NKR-P1 surface area via a ‘‘polar claw’’ mechanism. Polymorphisms in, and ablation of, the viral m12 protein and host NKR- P1B/C alleles impact NK cell responses in vivo. Thus, we identify the long-sought foreign ligand for this key immunoregulatory NKR family and reveal how it controls the evolutionary balance of immune recognition during host-pathogen interplay.

Human cytomegalovirus (HCMV) is the most common cause of viral infection acquired in utero. Even though the infection has been studied for several decades, immune determinants important for virus control and mechanisms of long-term sequelae caused by infection are still insufficiently characterized. Animal models of congenital HCMV infection provide unique opportunity to study various aspects of human disease. In this review, we summarize current knowledge on the role of immune system in congenital CMV infection, with emphasis on lessons learned from mouse model of congenital CMV infection.

NKp46/NCR1 is an activating NK-cell receptor implicated in the control of various viral and bacterial infections. Recent findings also suggest that it plays a role in shaping the adaptive immune response to pathogens. Using NCR1-deficient (NCR1(gfp/gfp)) mice, we provide evidence for the role of NCR1 in antibody response to mouse cytomegalovirus infection (MCMV). The absence of NCR1 resulted in impaired maturation, function and NK-cell migration to regional lymph nodes. In addition, CD4(+) T-cell activation and follicular helper T-cell (Tfh) generation were reduced, leading to inferior germinal center (GC) B-cell maturation. As a consequence, NCR1(gfp/gfp) mice produced lower amounts of MCMV-specific antibodies upon infection, which correlated with lower number of virus-specific antibody secreting cells in analyzed lymph nodes.

Cytomegaloviruses (CMVs) have dedicated a large portion of their genome towards immune evasion targeting many aspects of the host immune system, particularly NK cells. However, the host managed to cope with the infection by developing multiple mechanisms to recognize viral threat and counterattack it, thus illustrating never-ending evolutionary interplay between CMV and its host. In this review, we will focus on several mechanisms of NK cell evasion by mouse CMV (MCMV), the role of host inhibitory and activating Ly49 receptors involved in the virus control and acquisition of adaptive features by NK cells as a consequence of MCMV infection.

Development of an effective vaccine against human cytomegalovirus (HCMV) is a need of utmost medical importance. Generally, it is believed that a live attenuated vaccine would best provide protective immunity against this tenacious pathogen. Here, we propose a strategy for an HCMV vaccine that aims at the simultaneous activation of innate and adaptive immune responses. An HCMV strain expressing the host ligand ULBP2 for the NKG2D receptor was found to be susceptible to control by natural killer (NK) cells, and preserved the ability to stimulate HCMV-specific T cells. Infection with the ULBP2-expressing HCMV strain caused diminished cell surface levels of MHC class I molecules. While expression of the NKG2D ligand increased the cytolytic activity of NK cells, NKG2D engagement in CD8+ T cells provided co-stimulation and compensated for lower MHC class I expression. Altogether, our data indicate that triggering of both arms of the immune system is a promising approach applicable to the generation of a live attenuated HCMV vaccine., Author Summary Human cytomegalovirus (CMV) is a major cause of morbidity and mortality in congenitally infected newborns and immunocompromised individuals, indicating an utmost need for a vaccine to protect these vulnerable groups. Recent experimental studies in animal models, including non-human primates, have shown that attenuated CMVs trigger a potent immune response and are attractive vaccine candidates. However, an effective CMV vaccine is still not available. Here, we demonstrate that rational engineering of a live attenuated human CMV vaccine candidate is feasible. We equipped a CMV strain with an immunostimulatory molecule that is a ligand for an activating receptor present on both Natural Killer cells and CD8+ T cells. Moreover, we deleted several immunoevasins involved in downregulation of MHC class I molecules and of a ligand for Natural Killer cells in order to elicit stronger immune responses. In vitro assays using human immune cells and a first assessment in a humanized mouse model in vivo suggest that the generated CMV strain is attenuated and has the ability to induce a virus-specific immune response. Our study proposes this novel approach for the development of a rationally engineered CMV vaccine.

Regulatory T (Treg) cells dampen an exaggerated immune response to viral infections in order to avoid immunopathology. Cytomegaloviruses (CMVs) are herpesviruses usually causing asymptomatic infection in immunocompetent hosts and induce strong cellular immunity which provides protection against CMV disease. It remains unclear how these persistent viruses manage to avoid induction of immunopathology not only during the acute infection but also during life-long persistence and virus reactivation. This may be due to numerous viral immunoevasion strategies used to specifically modulate immune responses but also induction of Treg cells by CMV infection. Here we demonstrate that liver Treg cells are strongly induced in mice infected with murine CMV (MCMV). The depletion of Treg cells results in severe hepatitis and liver damage without alterations in the virus load. Moreover, liver Treg cells show a high expression of ST2, a cellular receptor for tissue alarmin IL-33, which is strongly upregulated in the liver of infected mice. We demonstrated that IL-33 signaling is crucial for Treg cell accumulation after MCMV infection and ST2-deficient mice show a more pronounced liver pathology and higher mortality compared to infected control mice. These results illustrate the importance of IL-33 in the suppressive function of liver Treg cells during CMV infection., Author summary Treg cells are crucial for immune homeostasis and for dampening immune response to several diseased conditions, including viral infections. Murine cytomegalovirus (MCMV) is a herpesvirus with pathogenic potential, so that early immune mechanisms are essential in controlling virus and protecting from virus-induced pathology. Studies on Foxp3+ Treg cells have revealed their inhibitory role on the early T cell response to MCMV infection and have suggested Treg cells as a target of MCMV’s immunoevasion mechanisms. Here we demonstrate that the number and activation status of liver Treg cells is strongly induced upon MCMV infection in order to protect the host from severe liver damage. They constitutively express high amounts of IL-33 receptor ST2 and their accumulation depends on IL-33, which is released as a tissue alarmin after the cell damage. For the first time, we show an immunoregulatory role of IL-33-dependent Treg cells in the liver of MCMV infected mice and their suppression of MCMV-induced immunopathology.

NK cells are innate lymphocytes that play a key role in the control of various viral infections. Recent studies indicate that NK cells may acquire some features of adaptive immune cells, including the formation of long-lived memory cells. A large and growing body of data indicates that NK cells regulate the adaptive immune response as well. The function and the activation status of NK cells are tightly regulated by signals induced by a broad range of inhibitory and activating cell surface receptors and cytokines released by other immune cells. Here, we review the function of mouse NK-cell receptors involved in virus control and in the regulation of the adaptive immune response. In addition, we discuss viral strategies used to evade NK-cell-mediated control during infection. Finally, the role of several activating Ly49 receptors specific for mouse cytomegalovirus (MCMV), as well as some controversial issues in the field, will be discussed.

Jonjic et al. show that inflammatory macrophages play an essential role in the control of murine CMV (MCMV) infection through a DNAM-1–PVR pathway., The poliovirus receptor (PVR) is a ubiquitously expressed glycoprotein involved in cellular adhesion and immune response. It engages the activating receptor DNAX accessory molecule (DNAM)-1, the inhibitory receptor TIGIT, and the CD96 receptor with both activating and inhibitory functions. Human cytomegalovirus (HCMV) down-regulates PVR expression, but the significance of this viral function in vivo remains unknown. Here, we demonstrate that mouse CMV (MCMV) also down-regulates the surface PVR. The m20.1 protein of MCMV retains PVR in the endoplasmic reticulum and promotes its degradation. A MCMV mutant lacking the PVR inhibitor was attenuated in normal mice but not in mice lacking DNAM-1. This attenuation was partially reversed by NK cell depletion, whereas the simultaneous depletion of mononuclear phagocytes abolished the virus control. This effect was associated with the increased expression of DNAM-1, whereas TIGIT and CD96 were absent on these cells. An increased level of proinflammatory cytokines in sera of mice infected with the virus lacking the m20.1 and an increased production of iNOS by inflammatory monocytes was observed. Blocking of CCL2 or the inhibition of iNOS significantly increased titer of the virus lacking m20.1. In this study, we have demonstrated that inflammatory monocytes, together with NK cells, are essential in the early control of CMV through the DNAM-1–PVR pathway.

MCMV-infected cells are recognized by multiple MHC class I–restricted Ly49-activating receptors in genetically distinct mouse strains., Recognition of mouse cytomegalovirus (MCMV)–infected cells by activating NK cell receptors was first described in the context of Ly49H, which confers resistance to C57BL/6 mice. We investigated the ability of other activating Ly49 receptors to recognize MCMV-infected cells in mice from various H-2 backgrounds. We observed that Ly49P1 from NOD/Ltj mice, Ly49L from BALB mice, and Ly49D2 from PWK/Pas mice respond to MCMV-infected cells in the context of H-2Dk and the viral protein m04/gp34. Recognition was also seen in the H-2d and/or H-2f contexts, depending on the Ly49 receptor examined, but never in H-2b. Furthermore, BALB.K (H-2k) mice showed reduced viral loads compared with their H-2d or H-2b congenic partners, a reduction which was dependent on interferon γ secretion by Ly49L+ NK cells early after infection. Adoptive transfer of Ly49L+, but not Ly49L−, NK cells significantly increased resistance against MCMV infection in neonate BALB.K mice. These results suggest that multiple activating Ly49 receptors participate in H-2–dependent recognition of MCMV infection, providing a common mechanism of NK cell–mediated resistance against viral infection.

Since their discovery, our understanding of NK cells has evolved from branding them marginal innate immunity cells to key players in anti-viral and anti-tumor immunity. Importance of NK cells in control of various viral infections is perhaps best illustrated by the existence of plethora of viral mechanisms aimed to modulate their function. These mechanisms include not only virally encoded immunoevasion proteins but also viral miRNA. Moreover, the evidence has been accumulated supporting the role of viral immunoevasion of NK cells in viral pathogenesis in vivo.

Human cytomegalovirus (HCMV) is a human pathogen that causes severe disease primarily in the immunocompromised or immunologically immature individual. To date, no vaccine is available. We describe use of a spread-deficient murine CMV (MCMV) as a novel approach for betaherpesvirus vaccination. To generate a spread-deficient MCMV, the conserved, essential gene M94 was deleted. Immunization with MCMV-Δ M94 is apathogenic and protective against wild-type challenge even in highly susceptible IFNαβR −/− mice. MCMV-Δ M94 was able to induce a robust CD4 + and CD8 + T-cell response as well as a neutralizing antibody response comparable to that induced by wild-type infection. Endothelial cells were identified as activators of CD8 + T cells in vivo . Thus, the vaccination with a spread-deficient betaherpesvirus is a safe and protective strategy and allows the linkage between cell tropism and immunogenicity. Furthermore, genomes of MCMV- ΔM94 were present in lungs 12 months after infection, revealing first-target cells as sites of genome maintenance.

Natural killer cell recognition of “missing self” contributes meaningfully to control of mouse cytomegalovirus infection in vivo., Cytomegaloviruses (CMVs) are renowned for interfering with the immune system of their hosts. To sidestep antigen presentation and destruction by CD8+ T cells, these viruses reduce expression of major histocompatibility complex class I (MHC I) molecules. However, this process sensitizes the virus-infected cells to natural killer (NK) cell–mediated killing via the “missing self” axis. Mouse cytomegalovirus (MCMV) uses m152 and m06 encoded proteins to inhibit surface expression of MHC I molecules. In addition, it encodes another protein, m04, which forms complexes with MHC I and escorts them to the cell surface. This mechanism is believed to prevent NK cell activation and killing by restoring the “self” signature and allowing the engagement of inhibitory Ly49 receptors on NK cells. Here we show that MCMV lacking m04 was attenuated in an NK cell– and MHC I–dependent manner. NK cell–mediated control of the infection was dependent on the presence of NK cell subsets expressing different inhibitory Ly49 receptors. In addition to providing evidence for immunoevasion strategies used by CMVs to avoid NK cell control via the missing-self pathway, our study is the first to demonstrate that missing self–dependent NK cell activation is biologically relevant in the protection against viral infection in vivo.

Natural killer (NK) cells are crucial in resistance to certain viral infections, but the mechanisms used to recognize infected cells remain largely unknown. Here, we show that the activating Ly49P receptor recognizes cells infected with mouse cytomegalovirus (MCMV) by a process that requires the presence of H2-D(k) and the MCMV m04 protein. Using H2 chimeras between H2-D(b) and -D(k), we demonstrate that the H2-D(k) peptide-binding platform is required for Ly49P recognition. We identified m04 as a viral component necessary for recognition using a panel of MCMV-deletion mutant viruses and complementation of m04-deletion mutant (Deltam04) virus infection. MA/My mice, which express Ly49P and H2-D(k), are resistant to MCMV ; however, infection with Deltam04 MCMV abrogates resistance. Depletion of NK cells in MA/My mice abrogates their resistance to wild-type MCMV infection, but does not significantly affect viral titers in mice infected with Deltam04 virus, implicating NK cells in host protection through m04-dependent recognition. These findings reveal a novel mechanism of major histocompatability complex class I-restricted recognition of virally infected cells by an activating NK cell receptor.

Host responses to viral infections involve complex interactions between cytokines and chemokines that provide communication signals resulting in the development of effective innate and adaptive immunity. Murine cytomegalovirus (MCMV) is a herpesvirus with pathogenic potential that can induce high levels of viral replication and modulate antiviral responses. Thus, early immune mechanisms are essential in controlling virus replication and protecting the host from virus- induced pathology. Interleukin-33 (IL-33) is a pro-inflammatory cytokine, expressed in the nucleus of nonhematopoetic cells, such as fibroblasts, endothelial and epithelial cells at barrier sites. It functions as an endogenous danger signal, alarmin, in response to tissue damage. Although its receptor, ST2, is expressed by many immune cells, it is highest on activated Th2 and mast cells where it participates in type 2 immunity and inflammation. However, signaling through ST2 exerts pleiotropic effects towards different cell types and it also amplifies CD8+ T cell (CTL) responses to viral infection, such as LCMV. In addition, it is necessary for IFN-γ production by NK cells. Here we show that ST2- deficient mice have weaker NK and CTL responses to MCMV infection than control wild-type mice. This indicates that IL-33 amplifies both NK and CTL responses to MCMV. In spite of this, ST2- deficient mice showed normal viral control, suggesting compensatory mechanism induced in absence of ST2 signaling. Moreover, ST2- deficient mice infected with high dose of MCMV showed higher death rate which is mediated by stronger liver pathology. This is a consequence of decreased infiltration od Treg cells and lower expression of anti-inflammatory IL-10 and TGF-β, compared to control mice. In the light of recent discovery of IL-33's importance for accumulation and maintenance of Treg cells in acute colitis model, our results suggest protective and homeostatic role of IL-33 signaling in inflammation induced by MCMV infection.

Herpesviruses form different gH/gL virion envelope glycoprotein complexes that serve as entry complexes for mediating viral cell-type tropism in vitro; their roles in vivo, however, remained speculative and can be addressed experimentally only in animal models. For murine cytomegalovirus two alternative gH/gL complexes, gH/gL/gO and gH/gL/MCK-2, have been identified. A limitation of studies on viral tropism in vivo has been the difficulty in distinguishing between infection initiation by viral entry into first-hit target cells and subsequent cell-to-cell spread within tissues. As a new strategy to dissect these two events, we used a gO-transcomplemented ΔgO mutant for providing the gH/gL/gO complex selectively for the initial entry step, while progeny virions lack gO in subsequent rounds of infection. Whereas gH/gL/gO proved to be critical for establishing infection by efficient entry into diverse cell types, including liver macrophages, endothelial cells, and hepatocytes, it was dispensable for intra-tissue spread. Notably, the salivary glands, the source of virus for host-to-host transmission, represent an exception in that entry into virus-producing cells did not strictly depend on either the gH/gL/gO or the gH/gL/MCK-2 complex. Only if both complexes were absent in gO and MCK-2 double-knockout virus, in vivo infection was abolished at all sites., Author Summary The role of viral glycoprotein entry complexes in viral tropism in vivo is a question central to understanding virus pathogenesis and transmission for any virus. Studies were limited by the difficulty in distinguishing between viral entry into first-hit target cells and subsequent cell-to-cell spread within tissues. Employing the murine cytomegalovirus entry complex gH/gL/gO as a paradigm for a generally applicable strategy to dissect these two events experimentally, we used a gO-transcomplemented ΔgO mutant for providing the complex exclusively for the initial cell entry step. In immunocompromised mice as a model for recipients of hematopoietic cell transplantation, our studies revealed an irreplaceable role for gH/gL/gO in initiating infection in host organs relevant to pathogenesis, whereas subsequent spread within tissues and infection of the salivary glands, the site relevant to virus host-to-host transmission, are double-secured by the entry complexes gH/gL/gO and gH/gL/MCK-2. As an important consequence, interventional strategies targeting only gO might be efficient in preventing organ manifestations after a primary viremia, whereas both gH/gL complexes need to be targeted for preventing intra-tissue spread of virus reactivated from latency within tissues as well as for preventing the salivary gland route of host-to-host transmission.

Recent advances in sequencing technology enabled us to perform comprehensive transcriptomic profiling of MCMV infection of MEFs and demonstrate that transcriptional profile and coding potential of herpesviruses have a much greater complexity than previously anticipated. Our analysis identified numerous novel spliced and unspliced MCMV transcripts, among which the most abundant viral transcripts are novel transcripts of unknown function. Among these, MAT (Most Abundant Transcript) is the most highly expressed throughout the infection. It has been shown earlier that MAT encodes a protein (Juranic Lisnic et al (2013) PLOS Pathogens) and contains a binding site for cellular microRNA miR-27 (Marcinowski et al (2012) PLOS Pathogens), making it a first reported viral transcript with coding and non-coding functions. We have recently detected an additional protein encoded by this enigmatic transcript, and here we report that deletion of the MCMV genomic region encoding MAT results in attenuation of the virus in vivo. However, the underlying mechanisms are not completely elucidated due to multiple functions MAT performs in the infected cells. On one hand, MAT deficiency results in failure to deplete cellular levels of miR-27, an important regulator of cell-cycle, while on the other MAT deletion mutants evade the detection by NK cells through activating Ly49 receptors P, L and D2. We also show that absence of MAT results in dramatic changes of host and viral peptides presented in the context of MHC I. In conclusion, not only does MAT provide an excellent example of the complex regulatory potential of herpesviral genes, where single transcripts exerts numerous and diverse functions, it also raises the question of how many new herpesviral genes, especially immune evasion genes, remain to be discovered.

Members of the α- and β-subfamily of herpesviridae encode glycoproteins that specifically bind to the Fc part of immunoglobulin (Ig)G. Plasma membrane resident herpesviral Fc receptors seem to prevent virus-specific IgG from activating antibody-dependent effector functions. We show that the mouse cytomegalovirus (MCMV) molecule fcr-1 promotes a rapid down-regulation of NKG2D ligands murine UL16-binding protein like transcript (MULT)-1 and H60 from the cell surface. Deletion of the m138/fcr-1 gene from the MCMV genome attenuates viral replication to natural killer (NK) cell response in an NKG2D-dependent manner in vivo. A distinct N-terminal module within the fcr-1 ectodomain in conjunction with the fcr-1 transmembrane domain was required to dispose MULT-1 to degradation in lysosomes. In contrast, down-modulation of H60 required the complete fcr-1 ectodomain, implying independent modes of fcr-1 interaction with the NKG2D ligands. The results establish a novel viral strategy for down-modulating NK cell responses and highlight the impressive diversity of Fc receptor functions.

Both human and mouse cytomegaloviruses (CMVs) encode proteins that inhibit the activation of NK cells by down-regulating cellular ligands for the activating NK cell receptor NKG2D. Up to now, three ligands for the NKG2D receptor, named RAE-1, H60, and MULT-1, have been identified in mice. The resistance of mouse strains to murine CMV (MCMV) infection is determined by their ability to generate an effective NK cell response. The MCMV genem152, a member of them145gene family, down-regulates the expression of RAE-1 in order to avoid NK cell control in vivo. Here we report that them155gene, another member of them145gene family, encodes a protein that interferes with the expression of H60 on the surfaces of infected cells. Deletion of them155gene leads to an only partial restoration of H60 expression on the cell surface, suggesting the involvement of another, so far unknown, viral inhibitor. In spite of this, anm155deletion mutant virus shows NK cell-dependent attenuation in vivo. The acquisition of endo-β-N-acetylglucosaminidase H resistance and the preserved half-life of H60 in MCMV-infected cells indicate that the m155-mediated effect must take place in a compartment after H60 exits from the ERGIC-cis-Golgi compartment.