A Viral Immunoevasin Controls Innate Immunity by Targeting the Prototypical Natural Killer Cell Receptor Family.

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 NKR-P1 (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.

Viral infection of the ovaries compromises pregnancy and reveals innate immune mechanisms protecting fertility.

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.

The herpesviral antagonist m152 reveals differential activation of STING-dependent IRF and NF-κB signaling and STING's dual role during MCMV infection.

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.

ChAdOx1-S adenoviral vector vaccine applied intranasally elicits superior mucosal immunity compared to the intramuscular route of vaccination.

COVID-19 vaccines prevent severe forms of the disease, but do not warrant complete protection against breakthrough infections. This could be due to suboptimal mucosal immunity at the site of virus entry, given that all currently approved vaccines are administered via the intramuscular route. In this study, we assessed humoral and cellular immune responses in BALB/c mice after intranasal and intramuscular immunization with adenoviral vector ChAdOx1-S expressing full-length Spike protein of SARS-CoV-2. We showed that both routes of vaccination induced a potent IgG antibody response, as well as robust neutralizing capacity, but intranasal vaccination elicited a superior IgA antibody titer in the sera and in the respiratory mucosa. Bronchoalveolar lavage from intranasally immunized mice efficiently neutralized SARS-CoV-2, which has not been the case in intramuscularly immunized group. Moreover, substantially higher percentages of epitope-specific CD8 T cells exhibiting a tissue resident phenotype were found in the lungs of intranasally immunized animals. Finally, both intranasal and intramuscular vaccination with ChAdOx1-S efficiently protected the mice after the challenge with recombinant herpesvirus expressing the Spike protein. Our results demonstrate that intranasal application of adenoviral vector ChAdOx1-S induces superior mucosal immunity and therefore could be a promising strategy for putting the COVID-19 pandemic under control.

Two murine cytomegalovirus microRNAs target the major viral immediate early 3 gene.

Human cytomegalovirus is responsible for morbidity and mortality in immune compromised patients and is the leading viral cause of congenital infection. Virus-encoded microRNAs (miRNAs) represent interesting targets for novel antiviral agents. While many cellular targets that augment productive infection have been identified in recent years, regulation of viral genes such as the major viral immediate early protein 72 (IE72) by hcmv-miR-UL112-1 may contribute to both the establishment and the maintenance of latent infection. We employed photoactivated ribonucleotide-enhanced individual nucleotide resolution crosslinking (PAR-iCLIP) to identify murine cytomegalovirus (MCMV) miRNA targets during lytic infection. While the PAR-iCLIP data were of insufficient quality to obtain a comprehensive list of cellular and viral miRNA targets, the most prominent PAR-iCLIP peak in the MCMV genome mapped to the 3' untranslated region of the major viral immediate early 3 (ie3) transcript. We show that this results from two closely positioned binding sites for the abundant MCMV miRNAs miR-M23-2-3p and miR-m01-2-3p. Their pre-expression significantly impaired viral plaque formation. However, mutation of the respective binding sites did not alter viral fitness during acute or subacute infection in vivo. Furthermore, no differences in the induction of virus-specific CD8+ T cells were observed. Future studies will probably need to go beyond studying immunocompetent laboratory mice housed in pathogen-free conditions to reveal the functional relevance of viral miRNA-mediated regulation of key viral immediate early genes.

Mucosal CD8+ T cell responses induced by an MCMV based vaccine vector confer protection against influenza challenge.

Cytomegalovirus (CMV) is a ubiquitous ß-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.

Mouse Cytomegalovirus m153 Protein Stabilizes Expression of the Inhibitory NKR-P1B Ligand Clr-b.

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 Clec2d) 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 m153-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 Clec2d 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 in vivo, 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.IMPORTANCE 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.

Brain-resident memory CD8+ T cells induced by congenital CMV infection prevent brain pathology and virus reactivation.

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.

IL-33/ST2 pathway drives regulatory T cell dependent suppression of liver damage upon cytomegalovirus infection.

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.

Role of antibodies in confining cytomegalovirus after reactivation from latency: three decades' résumé.

Cytomegaloviruses (CMVs) are highly prevalent herpesviruses, characterized by strict species specificity and the ability to establish non-productive latent infection from which reactivation can occur. Reactivation of latent human CMV (HCMV) represents one of the most important clinical challenges in transplant recipients secondary to the strong immunosuppression. In addition, HCMV is the major viral cause of congenital infection with severe sequelae including brain damage. The accumulated evidence clearly shows that cellular immunity plays a major role in the control of primary CMV infection as well as establishment and maintenance of latency. However, the efficiency of antiviral antibodies in virus control, particularly in prevention of congenital infection and virus reactivation from latency in immunosuppressed hosts, is much less understood. Because of a strict species specificity of HCMV, the role of antibodies in controlling CMV disease has been addressed using murine CMV (MCMV) as a model. Here, we review and discuss the role played by the antiviral antibody response during CMV infections with emphasis on latency and reactivation not only in the MCMV model, but also in relevant clinical settings. We provide evidence to conclude that antiviral antibodies do not prevent the initiating molecular event of virus reactivation from latency but operate by preventing intra-organ spread and inter-organ dissemination of recurrent virus.

CD4 T cells are required for maintenance of CD8 TRM cells and virus control in the brain of MCMV-infected newborn mice.

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.

NCR1-deficiency diminishes the generation of protective murine cytomegalovirus antibodies by limiting follicular helper T-cell maturation.

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 (NCR1gfp/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, NCR1gfp/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.

Cytomegalovirus vector expressing RAE-1γ induces enhanced anti-tumor capacity of murine CD8+ T cells.

Designing CD8+ T-cell vaccines, which would provide protection against tumors is still considered a great challenge in immunotherapy. Here we show the robust potential of 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. CMV vector expressing RAE-1γ 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.

Activation of Innate and Adaptive Immunity by a Recombinant Human Cytomegalovirus Strain Expressing an NKG2D Ligand.

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.

Altered NK cell development and enhanced NK cell-mediated resistance to mouse cytomegalovirus in NKG2D-deficient mice.

NKG2D is a potent activating receptor on natural killer (NK) cells and acts as a molecular sensor for stressed cells expressing NKG2D ligands such as infected or tumor-transformed cells. Although NKG2D is expressed on NK cell precursors, its role in NK cell development is not known. We have generated NKG2D-deficient mice by targeting the Klrk1 locus. Here we provide evidence for an important regulatory role of NKG2D in the development of NK cells. The absence of NKG2D caused faster division of NK cells, perturbation in size of some NK cell subpopulations, and their augmented sensitivity to apoptosis. As expected, Klrk1(-/-) NK cells are less responsive to tumor targets expressing NKG2D ligands. Klrk1(-/-) mice, however, showed an enhanced NK cell-mediated resistance to mouse cytomegalovirus infection as a consequence of NK cell dysregulation. Altogether, these findings provide evidence for regulatory function of NKG2D in NK cell physiology.

Non-redundant and redundant roles of cytomegalovirus gH/gL complexes in host organ entry and intra-tissue spread.

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.

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

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 specific NK cell response in concert with perforin prevents CD8(+) T cell-mediated immunopathology after mouse cytomegalovirus infection.

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.

The evolutionary arms race between NK cells and viruses: who gets the short end of the stick?

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.

Viral inhibition of BAK promotes murine cytomegalovirus dissemination to salivary glands.

Apoptosis induction is an important host defense mechanism to control viral infection, which is antagonized by viral proteins. Murine cytomegalovirus m41.1 encodes a viral inhibitor of BAK oligomerization (vIBO) that blocks the mitochondrial apoptosis mediator BAK. However, its importance for viral fitness in vivo has not been investigated. Here, we show that an m41.1-deficient virus attains reduced titers in salivary glands of wild-type but not Bak1(-/-) mice, indicating a requirement of BAK inhibition for optimal dissemination in vivo.