Mouse Cytomegalovirus Research Articles

Tegument Protein pp150 Sequence-Specific Peptide Blocks Cytomegalovirus Infection.

Human cytomegalovirus (HCMV) tegument protein pp150 is essential for the completion of the final steps in virion maturation. Earlier studies indicated that three pp150nt (N-terminal one-third of pp150) conformers cluster on each triplex (Tri1, Tri2A and Tri2B), and extend towards small capsid proteins atop nearby major capsid proteins, forming a net-like layer of tegument densities that enmesh and stabilize HCMV capsids. Based on this atomic detail, we designed several peptides targeting pp150nt. Our data show significant reduction in virus growth upon treatment with one of these peptides (pep-CR2) with an IC50 of 1.33 μM and no significant impact on cell viability. Based on 3D modeling, pep-CR2 specifically interferes with the pp150–capsid binding interface. Cells pre-treated with pep-CR2 and infected with HCMV sequester pp150 in the nucleus, indicating a mechanistic disruption of pp150 loading onto capsids and subsequent nuclear egress. Furthermore, pep-CR2 effectively inhibits mouse cytomegalovirus (MCMV) infection in cell culture, paving the way for future animal testing. Combined, these results indicate that CR2 of pp150 is amenable to targeting by a peptide inhibitor, and can be developed into an effective antiviral.

Species-Specific Inhibition of Necroptosis by HCMV UL36.

Viral infection activates cellular antiviral defenses including programmed cell death (PCD). Many viruses, particularly those of the Herpesviridae family, encode cell death inhibitors that antagonize different forms of PCD. While some viral inhibitors are broadly active in cells of different species, others have species-specific functions, probably reflecting the co-evolution of the herpesviruses with their respective hosts. Human cytomegalovirus (HCMV) protein UL36 is a dual cell death pathway inhibitor. It blocks death receptor-dependent apoptosis by inhibiting caspase-8 activation, and necroptosis by binding to the mixed lineage kinase domain-like (MLKL) protein and inducing its degradation. While UL36 has been shown to inhibit apoptosis in human and murine cells, the specificity of its necroptosis-inhibiting function has not been investigated. Here we show that UL36 interacts with both human and murine MLKL, but has a higher affinity for human MLKL. When expressed by a recombinant mouse cytomegalovirus (MCMV), UL36 caused a modest reduction of murine MLKL levels but did not inhibit necroptosis in murine cells. These data suggest that UL36 inhibits necroptosis, but not apoptosis, in a species-specific manner, similar to ICP6 of herpes simplex virus type 1 and MC159 of molluscum contagiosum virus. Species-specific necroptosis inhibition might contribute to the narrow host range of these viruses.

A Live Olfactory Mouse Cytomegalovirus Vaccine, Attenuated for Systemic Spread, Protects against Superinfection.

Vaccination against the betaherpesvirus, human cytomegalovirus (HCMV) is a public health goal. However, HCMV has proved difficult to vaccinate against. Vaccination against single HCMV determinants has not worked, suggesting that immunity to a wider antigenic profile may be required. Live attenuated vaccines provide the best prospects for protection, but the question remains as to how to balance vaccine virulence with safety. Animal models of HCMV infection provide insights into identifying targets for virus attenuation and understanding how host immunity blocks natural, mucosal infection. Here, we evaluated the vaccine potential of a mouse cytomegalovirus (MCMV) vaccine deleted of a viral G protein-coupled receptor (GPCR), designated M33, that renders it attenuated for systemic spread. A single noninvasive olfactory ΔM33 MCMV vaccine replicated locally, but as a result of the loss of the M33 GPCR, it failed to spread systemically and was attenuated for latent infection. Vaccination did not prevent host entry of a superinfecting MCMV but spread from the mucosa was blocked. This approach to vaccine design may provide a viable alternative for a safe and effective betaherpesvirus vaccine. IMPORTANCE Human cytomegalovirus (HCMV) is the most common cause of congenital infection for which a vaccine is not yet available. Subunit vaccine candidates have failed to achieve licensure. A live HCMV vaccine may prove more efficacious, but it faces safety hurdles which include its propensity to persist and to establish latency. Understanding how pathogens infect guide rational vaccine design. However, HCMV infections are asymptomatic and thus difficult to capture. Animal models of experimental infection provide insight. Here, we investigated the vaccine potential of a mouse cytomegalovirus (MCMV) attenuated for systemic spread and latency. We used olfactory vaccination and virus challenge to mimic its natural acquisition. We provide proof of concept that a single olfactory MCMV that is deficient in systemic spread can protect against wild-type MCMV superinfection and dissemination. This approach of deleting functional counterpart genes in HCMV may provide safe and effective vaccination against congenital HCMV disease.

Nedd8-Activating Enzyme Is a Druggable Host Dependency Factor of Human and Mouse Cytomegalovirus.

Human cytomegalovirus causes diseases in individuals with insufficient immunity. Cytomegaloviruses exploit the ubiquitin proteasome pathway to manipulate the proteome of infected cells. The proteasome degrades ubiquitinated proteins. The family of cullin RING ubiquitin ligases (CRL) regulates the stability of numerous important proteins. If the cullin within the CRL is modified with Nedd8 (“neddylated”), the CRL is enzymatically active, while CRLs lacking Nedd8 modifications are inactive. The Nedd8-activating enzyme (NAE) is indispensable for neddylation. By binding to NAE and inhibiting neddylation, the drug MLN4924 (pevonedistat) causes CRL inactivation and stabilization of CRL target proteins. We showed that MLN4924 elicits potent antiviral activity against cytomegaloviruses, suggesting that NAE might be a druggable host dependency factor (HDF). However, MLN4924 is a nucleoside analog related to AMP, and the antiviral activity of MLN4924 may have been influenced by off-target effects in addition to NAE inhibition. To test if NAE is indeed an HDF, we assessed the novel NAE inhibitor TAS4464 and observed potent antiviral activity against mouse and human cytomegalovirus. Additionally, we raised an MLN4924-resistant cell clone and showed that MLN4924 as well as TAS4464 lose their antiviral activity in these cells. Our results indicate that NAE, the neddylation process, and CRLs are druggable HDFs of cytomegaloviruses.

Cytomegalovirus subverts macrophage identity

Cytomegaloviruses (CMVs) have co-evolved with their mammalian hosts for millions of years, leading to remarkable host specificity and high infection prevalence. Macrophages, which already populate barrier tissues in the embryo, are the predominant immune cells at potential CMV entry sites. Here we show that, upon CMV infection, macrophages undergo a morphological, immunophenotypic, and metabolic transformation process with features of stemness, altered migration, enhanced invasiveness, and provision of the cell cycle machinery for viral proliferation. This complex process depends on Wnt signaling and the transcription factor ZEB1. In pulmonary infection, mouse CMV primarily targets and reprograms alveolar macrophages, which alters lung physiology and facilitates primary CMV and secondary bacterial infection by attenuating the inflammatory response. Thus, CMV profoundly perturbs macrophage identity beyond established limits of plasticity and rewires specific differentiation processes, allowing viral spread and impairing innate tissue immunity.

Cytomegalovirus Infection and Inflammation in Developing Brain.

Human cytomegalovirus (HCMV) is a highly prevalent herpesvirus that can cause severe disease in immunocompromised individuals and immunologically immature fetuses and newborns. Most infected newborns are able to resolve the infection without developing sequelae. However, in severe cases, congenital HCMV infection can result in life-threatening pathologies and permanent damage of organ systems that possess a low regenerative capacity. Despite the severity of the problem, HCMV infection of the central nervous system (CNS) remains inadequately characterized to date. Cytomegaloviruses (CMVs) show strict species specificity, limiting the use of HCMV in experimental animals. Infection following intraperitoneal administration of mouse cytomegalovirus (MCMV) into newborn mice efficiently recapitulates many aspects of congenital HCMV infection in CNS. Upon entering the CNS, CMV targets all resident brain cells, consequently leading to the development of widespread histopathology and inflammation. Effector functions from both resident cells and infiltrating immune cells efficiently resolve acute MCMV infection in the CNS. However, host-mediated inflammatory factors can also mediate the development of immunopathologies during CMV infection of the brain. Here, we provide an overview of the cytomegalovirus infection in the brain, local immune response to infection, and mechanisms leading to CNS sequelae.

Memory CD8 T Cells Generated by Cytomegalovirus Vaccine Vector Expressing NKG2D Ligand Have Effector-Like Phenotype and Distinct Functional Features.

Viral vectors have emerged as a promising alternative to classical vaccines due to their great potential for induction of a potent cellular and humoral immunity. Cytomegalovirus (CMV) is an attractive vaccine vector due to its large genome with many non-essential immunoregulatory genes that can be easily manipulated to modify the immune response. CMV generates a strong antigen-specific CD8 T cell response with a gradual accumulation of these cells in the process called memory inflation. In our previous work, we have constructed a mouse CMV vector expressing NKG2D ligand RAE-1γ in place of its viral inhibitor m152 (RAE-1γMCMV), which proved to be highly attenuated in vivo. Despite attenuation, RAE-1γMCMV induced a substantially stronger CD8 T cell response to vectored antigen than the control vector and provided superior protection against bacterial and tumor challenge. In the present study, we confirmed the enhanced protective capacity of RAE-1γMCMV as a tumor vaccine vector and determined the phenotypical and functional characteristics of memory CD8 T cells induced by the RAE-1γ expressing MCMV. RNAseq data revealed higher transcription of numerous genes associated with effector-like CD8 T cell phenotype in RAE-1γMCMV immunized mice. CD8 T cells primed with RAE-1γMCMV were enriched in TCF1 negative population, with higher expression of KLRG1 and lower expression of CD127, CD27, and Eomes. These phenotypical differences were associated with distinct functional features as cells primed with RAE-1γMCMV showed inferior cytokine-producing abilities but comparable cytotoxic potential. After adoptive transfer into naive hosts, OT-1 cells induced with both RAE-1γMCMV and the control vector were equally efficient in rejecting established tumors, suggesting the context of latent infection and cell numbers as important determinants of enhanced anti-tumor response following RAE-1γMCMV vaccination. Overall, our results shed new light on the phenotypical and functional distinctness of memory CD8 T cells induced with CMV vector expressing cellular ligand for the NKG2D receptor.

NK/ILC1 cells mediate neuroinflammation and brain pathology following congenital CMV infection.

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.

Prophylactic and therapeutic HBV vaccination by an HBs-expressing cytomegalovirus vector lacking an interferon antagonist in mice.

Cytomegalovirus (CMV)-based vaccines show promising effects against chronic infections in nonhuman primates. Therefore, we examined the potential of hepatitis B virus (HBV) vaccines based on mouse CMV (MCMV) vectors expressing the small HBsAg. Immunological consequences of vaccine virus attenuation were addressed by either replacing the dispensable gene m157 ("MCMV-HBsȍ) or the gene M27 ("ΔM27-HBs"), the latter encodes a potent IFN antagonist targeting the transcription factor STAT2. M27 was chosen, since human CMV encodes an analogous gene product, which also induced proteasomal STAT2 degradation by exploiting Cullin RING ubiquitin ligases. Vaccinated mice were challenged with HBV through hydrodynamic injection. MCMV-HBs and ΔM27-HBs vaccination achieved accelerated HBV clearance in serum and liver as well as robust HBV-specific CD8+ T-cell responses. When we explored the therapeutic potential of MCMV-based vaccines, especially the combination of ΔM27-HBs prime and DNA boost vaccination resulted in increased intrahepatic HBs-specific CD8+ T-cell responses and HBV clearance in persistently infected mice. Our results demonstrated that vaccines based on a replication competent MCMV attenuated through the deletion of an IFN antagonist targeting STAT2 elicit robust anti-HBV immune responses and mediate HBV clearance in mice in prophylactic and therapeutic immunization regimes.

Artemisinins target the intermediate filament protein vimentin for human cytomegalovirus inhibition

The antimalarial agents artemisinins inhibit cytomegalovirus (CMV) in vitro and in vivo, but their target(s) has been elusive. Using a biotin-labeled artemisinin, we identified the intermediate filament protein vimentin as an artemisinin target, validated by detailed biochemical and biological assays. We provide insights into the dynamic and unique modulation of vimentin, depending on the stage of human CMV (HCMV) replication. In vitro, HCMV entry and viral progeny are reduced in vimentin-deficient fibroblasts, compared with control cells. Similarly, mouse CMV (MCMV) replication in vimentin knockout mice is significantly reduced compared with controls in vivo, confirming the requirement of vimentin for establishment of infection. Early after HCMV infection of human foreskin fibroblasts vimentin level is stable, but as infection proceeds, vimentin is destabilized, concurrent with its phosphorylation and virus-induced calpain activity. Intriguingly, in vimentin-overexpressing cells, HCMV infection is reduced compared with control cells. Binding of artesunate, an artemisinin monomer, to vimentin prevents virus-induced vimentin degradation, decreasing vimentin phosphorylation at Ser-55 and Ser-83 and resisting calpain digestion. In vimentin-deficient fibroblasts, the anti-HCMV activity of artesunate is reduced compared with controls. In summary, an intact and stable vimentin network is important for the initiation of HCMV replication but hinders its completion. Artesunate binding to vimentin early during infection stabilizes it and antagonizes subsequent HCMV-mediated vimentin destabilization, thus suppressing HCMV replication. Our target discovery should enable the identification of vimentin-binding sites and compound moieties for binding.

Murine Cytomegalovirus M25 Proteins Sequester the Tumor Suppressor Protein p53 in Nuclear Accumulations.

To ensure productive infection, herpesviruses utilize tegument proteins and nonstructural regulatory proteins to counteract cellular defense mechanisms and to reprogram cellular pathways. The M25 proteins of mouse cytomegalovirus (MCMV) belong to the betaherpesvirus UL25 gene family that encodes viral proteins implicated with regulatory functions. Through affinity purification and mass spectrometric analysis, we discovered the tumor suppressor protein p53 as a host factor interacting with the M25 proteins. M25-p53 interaction in infected and transfected cells was confirmed by coimmunoprecipitation. Moreover, the proteins colocalized in nuclear dot-like structures upon both infection and inducible expression of the two M25 isoforms. p53 accumulated in wild-type MCMV-infected cells, while this did not occur upon infection with a mutant lacking the M25 gene. Both M25 proteins were able to mediate the effect, identifying them as the first CMV proteins responsible for p53 accumulation during infection. Interaction with M25 proteins led to substantial prolongation of the half-life of p53. In contrast to the higher abundance of the p53 protein in wild-type MCMV-infected cells, the transcript levels of the prominent p53 target genes Cdkn1a and Mdm2 were diminished compared to cells infected with the ΔM25 mutant, and this was associated with reduced binding of p53 to responsive elements within the respective promoters. Notably, the productivity of the M25 deletion mutant was partially rescued on p53-negative fibroblasts. We propose that the MCMV M25 proteins sequester p53 molecules in the nucleus of infected cells, reducing their availability for activating a subset of p53-regulated genes, thereby dampening the antiviral role of p53.IMPORTANCE Host cells use a number of factors to defend against viral infection. Viruses are, however, in an arms race with their host cells to overcome these defense mechanisms. The tumor suppressor protein p53 is an important sensor of cell stress induced by oncogenic insults or viral infections, which upon activation induces various pathways to ensure the integrity of cells. Viruses have to counteract many functions of p53, but complex DNA viruses such as cytomegaloviruses may also utilize some p53 functions for their own benefit. In this study, we discovered that the M25 proteins of mouse cytomegalovirus interact with p53 and mediate its accumulation during infection. Interaction with the M25 proteins sequesters p53 molecules in nuclear dot-like structures, limiting their availability for activation of a subset of p53-regulated target genes. Understanding the interaction between viral proteins and p53 may allow to develop new therapeutic strategies against cytomegalovirus and other viruses.

Tetramer Immunization and Selection Followed by CELLISA Screening to Generate Monoclonal Antibodies against the Mouse Cytomegalovirus m12 Immunoevasin.

The generation of reliable mAb of unique and desired specificities serves as a valuable technology to study protein expression and function. However, standard approaches to mAb generation usually involve large-scale protein purification and intensive screening. In this study, we describe an optimized high-throughput proof-of-principle method for the expanded generation, enrichment, and screening of mouse hybridomas secreting mAb specific for a protein of interest. Briefly, we demonstrate that small amounts of a biotinylated protein of interest can be used to generate tetramers for use as prime-boost immunogens, followed by selective enrichment of Ag-specific B cells by magnetic sorting using the same tetramers prior to hybridoma generation. This serves two purposes: 1) to effectively expand both low- and high-affinity B cells specific for the antigenic bait during immunization and 2) to minimize subsequent laborious hybridoma efforts by positive selection of Ag-specific, Ab-secreting cells prior to hybridoma fusion and validation screening. Finally, we employ a rapid and inexpensive screening technology, CELLISA, a high-throughput validation method that uses a chimeric Ag fused to the CD3ζ signaling domain expressed on enzyme-generating reporter cells; these reporters can detect specific mAb in hybridoma supernatants via plate-bound Ab-capture arrays, thereby easing screening. Using this strategy, we generated and characterized novel mouse mAb specific for a viral immunoevasin, the mouse CMV m12 protein, and suggest that these mAb may protect mice from CMV infection via passive immunity.

Airborne Aerosolized Mouse Cytomegalovirus From Common Otolaryngology Procedures: Implications for COVID-19 Infection.

ObjectivesTo determine whether common otolaryngology procedures generate viable aerosolized virus through a murine cytomegalovirus (mCMV) model for infection.Study DesignmCMV model of infection.SettingUniversity of Utah laboratory.MethodsThree-day-old BALB/c mice were inoculated with mCMV or saline. Five days later, each mouse underwent drilling, microdebrider, coblation, and electrocautery procedures. Particle size distribution and PM2.5 (particulate matter <2.5 µm) concentration were determined with a scanning mobility particle sizer and an aerosol particle sizer in the range of 15 nm to 32 µm. Aerosolized samples from these procedures were collected with an Aerosol Devices BioSpot sampler for viral titer based on polymerase chain reaction and for viable virus through viral culture.ResultsAs compared with the background aerosol concentrations, coblation and electrocautery showed statistically significant increases in airborne aerosols (Tukey-adjusted P value <.040), while microdebrider and drilling at 30,000 rpm did not (.870 < Tukey-adjusted P value < .930). We identified viral DNA in samples from coblation and drilling procedures, although we did not identify viable viruses in aerosol samples from any of the 4 procedures.ConclusionCoblation and electrocautery procedures generate >100-fold increases in aerosol concentrations over background; only coblation and drilling produce aerosolized viral DNA. The high concentration of aerosols from coblation and electrocautery suggests the need for appropriate safeguards against particle exposure to health care workers. The presence of viral DNA from drilling and coblation procedures warrants the need for appropriate protection against droplet and aerosol exposure.

Coordinated Viral Control by Cytotoxic Lymphocytes Ensures Optimal Adaptive NK Cell Responses

SUMMARYNatural killer (NK) cells play a critical role in controlling viral infections, coordinating the response of innate and adaptive immune systems. They also possess certain features of adaptive lymphocytes, such as undergoing clonal proliferation. However, it is not known whether this adaptive NK cell response can be modulated by other lymphocytes during viral exposure. Here, we show that the clonal expansion of NK cells during mouse cytomegalovirus infection is severely blunted in the absence of cytotoxic CD8+ T cells. This correlates with higher viral burden and an increased pro-inflammatory milieu, which maintains NK cells in a hyper-activated state. Antiviral therapy rescues NK cell expansion in the absence of CD8+ T cells, suggesting that high viral loads have detrimental effects on adaptive NK cell responses. Altogether, our data support a mechanism whereby cytotoxic innate and adaptive lymphocytes cooperate to ensure viral clearance and the establishment of robust clonal NK cell responses.

The activation trajectory of plasmacytoid dendritic cells in vivo during a viral infection.

Plasmacytoid dendritic cells (pDCs) are a major source of type I interferon (IFN-I). What other functions pDCs exert in vivo during viral infections is controversial and more studies are needed to understand their orchestration. Here, we characterize in-depth and link pDC activation states in animals infected by mouse cytomegalovirus, by combining Ifnb1 reporter mice with flow cytometry, single-cell RNA sequencing, confocal microscopy and a cognate CD4 T cell activation assay. We show that IFN-I production and T cell activation were performed by the same pDC, but sequentially in time and in different micro-anatomical locations. In addition, we show that pDC commitment to IFN-I production was marked early on by their downregulation of LIFR and promoted by cell-intrinsic TNF signaling. We propose a novel model of how individual pDCs are endowed to exert different functions in vivo during a viral infection in a manner tightly orchestrated in time and space.

Cutting Edge: STAT1-Mediated Epigenetic Control of Rsad2 Promotes Clonal Expansion of Antiviral NK Cells.

NK cells represent a cellular component of innate immunity but possess features of adaptive immunity, including clonal expansion and establishment of long-lived memory following infection. During mouse CMV (MCMV) infection, we observed Rsad2 (which encodes Viperin) to be among the most highly induced IFN stimulatory genes in activated NK cells, correlating with increased chromatin accessibility at the Rsad2 gene locus. Furthermore, in NK cells stimulated with IFN-α, the promoter region of Rsad2 was enriched for STAT1 binding and the permissive histone mark H3K4me3. IFN-αR- and STAT1-deficient NK cells showed an impairment of Rsad2 induction and chromatin accessibility during MCMV infection. Finally, Rsad2-deficient NK cells were defective in clonal expansion and memory formation following exposure to MCMV, in part because of greater apoptosis. Thus, our study reveals a critical mechanism of STAT1-mediated epigenetic control of Rsad2 to promote the adaptive behavior of NK cells during viral infection.

Differential cleavage of viral polypeptides by allotypic variants of granzyme B skews immunity to mouse cytomegalovirus

We investigated the molecular basis for the remarkably different survival outcomes of mice expressing different alloforms of the pro-apoptotic serine protease granzyme B to mouse cytomegalovirus infection. Whereas C57BL/6 mice homozygous for granzyme BP (GzmBP/P) raise cytotoxic T lymphocytes that efficiently kill infected cells, those of C57BL/6 mice congenic for the outbred allele (GzmBW/W) fail to kill MCMV-infected cells and died from uncontrolled hepatocyte infection and acute liver failure. We identified subtle differences in how GzmBP and GzmBW activate cell death signalling - both alloforms predominantly activated pro-caspases directly, and cleaved pro-apoptotic Bid poorly. Consequently, neither alloform initiated mitochondrial outer membrane permeabilization, or was blocked by Bcl-2, Bcl-XL or co-expression of MCMV proteins M38.5/M41.1, which together stabilize mitochondria by sequestering Bak/Bax. Remarkably, mass spectrometric analysis of proteins from MCMV-infected primary mouse embryonic fibroblasts identified 13 cleavage sites in nine viral proteins (M18, M25, M28, M45, M80, M98, M102, M155, M164) that were cleaved >20-fold more efficiently by either GzmBP or GzmBW. Notably, M18, M28, M45, M80, M98, M102 and M164 were cleaved 20- >100-fold more efficiently by GzmBW, and so, would persist in infected cells targeted by CTLs from GzmBP/P mice. Conversely, M155 was cleaved >100-fold more efficiently by GzmBP, and would persist in cells targeted by CTLs of GzmBW/W mice. M25 was cleaved efficiently by both proteases, but at different sites. We conclude that different susceptibility to MCMV does not result from skewed endogenous cell death pathways, but rather, to as yet uncharacterised MCMV-intrinsic pathways that ultimately inhibit granzyme B-induced cell death.

NK Cell-Mediated Recall Responses: Memory-Like, Adaptive, or Antigen-Specific?

Mounting experimental evidence hints to an import role for natural killer (NK) cells in adaptive immune responses to pathogens. NK cells with adaptive features are heterogeneous and belong to different subsets according to their phenotype as well as the nature of their adaptive recall reactions. Three types of adaptive NK cell responses have been described: (i) NK cells with long-lived memory of multiple different haptens and viral antigens were described in murine liver tissue with a possible human counterpart; (ii) infection of human and mouse cytomegalovirus is associated with an expansion of NKG2C+ and Ly49H+ NK cells, respectively, that selectively recognize CMV-encoded peptides thereby facilitating recall responses; (iii) cytokine-stimulated NK cells respond to different stimuli with enhanced production of IFN-γ after re-stimulation. These exciting findings not only support the idea of NK cells with adaptive features, but define a novel field of harnessing memory NK cell subsets for therapeutic strategies.

Mouse Cytomegalovirus M34 Encodes a Non-essential, Nuclear, Early-Late Expressed Protein Required for Efficient Viral Replication.

Human cytomegalovirus (HCMV) is a prototypic betaherpesvirus which causes severe manifestations in individuals with impaired or immature immunity. To investigate cytomegalovirus-induced pathogenesis and virus-specific immune responses, mouse cytomegalovirus (MCMV) infections in mice are employed as accepted small animal model. MCMV and HCMV share co-linear genomes and encode several homologous proteins. Due to the size and complexity of CMV genomes, the molecular functions of numerous cytomegaloviral gene products remain to be elucidated. While the essential nature of viral genes highlights their biological relevance, it renders functional studies particularly cumbersome by precluding experiments in the infection context. The HCMV-encoded protein pUL34 binds the HCMV genome and regulates viral gene expression (e.g., of US3). Several groups provided compelling evidence that UL34 is essential for HCMV replication. MCMV encodes the homologous protein pM34 (34% identical and 55% similar). Based on unsuccessful attempts to reconstitute M34-deficient virus from a bacterial artificial chromosome (BAC), M34 was previously classified as essential for MCMV replication. To characterize pM34 during viral infection, we engineered and analyzed an MCMV mutant expressing an HA-epitope-tagged pM34 which was expressed with early-late kinetics and localized in the nucleus. Additionally, we generated an M34-deficient (“ΔM34”) MCMV-BAC by replacing the entire M34 coding sequence by a kanamycin resistance cassette. The deletion of M34 was confirmed by Southern blot and PCR. Unexpectedly, we could reconstitute replicating ΔM34-MCMV upon transfection of the BAC DNA into mouse embryonic fibroblasts. The absence of M34 from the genome of the replicating ΔM34-MCMV was also confirmed. Accordingly, a ΔM34-MCMV, in which the kanamycin cassette was excised by frt/Flp-mediated recombination, was also replication competent. In order to corroborate the absence of pM34 protein, the M34 deletion was recapitulated on the background of M34HA, which yielded replicating virus devoid of detectable pM34HA protein. The replication of MCMVs lacking M34 was found to be 10- to 100-fold reduced as compared to wt-MCMV which might explain previous unsuccessful reconstitution attempts conducted by others. Taken together, our findings reveal that MCMV remains replication competent despite the absence of M34, enabling functional studies in the infection context.

Continuous generation of effector (Teff) and effector memory (Tem) T cells with mouse cytomegalovirus (MCMV) vaccination to prolong malaria immunity

Abstract Immunity to Plasmodium infection or vaccination decays without continuous exposure. This decay correlates with loss of malaria-specific T cells, not antibody. We recently demonstrated that Teff protect better than memory T cells, and that Ifng+ Teff are responsible for the protection provided by persistent P. chabaudi infection. Based on this data, we have reverse-engineered a vaccine strategy that first induces a long-lived antibody response, and then boosts the Teff and Tem response to prolong protection. We chose live vaccination with “infection and drug cure”, which generates protection that decays over 200 days. The boost uses MCMV as it is an excellent T cell-inducing vector with promise against SIV, tuberculosis and liver-stage malaria. First, we observed that MCMV infection strongly reduced parasite growth, as previously shown for Leishmania, suggesting a useful adjuvant effect. To promote continuous induction of specific T cells, we generated MCMV expressing the P. chabaudi T cell epitope B5. Vaccination with MCMV-B5 activated both adoptively transferred B5 TCR Tg T cells and MCMV-specific CD4 T cells, generating protective Teff and Tem phenotypes for over 2.5 months. Importantly, we show that boosting with the MCMV vector prolongs protection generated by the live vaccine, as shown by better protection from re-infection at day 200. We are currently investigating the contribution of activated Plasmodium-specific T cells in prolonging protective immunity by MCMV-B5, and mechanisms of protection. Our findings suggest that chronic vaccination can redress the problem of short-lived protection to malaria infection and vaccines, which would be beneficial for any efficacious but short-term vaccine strategy.