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(1) Background: Infection with human cytomegalovirus (HCMV) leads to the production and release of subviral particles, termed Dense Bodies (DB). They are enclosed by a membrane resembling the viral envelope. This membrane mediates the entrance of DBs into cells in a way that is comparable to virus infection. HCMV attachment and entry trigger the induction of interferon synthesis and secretion, and the subsequent expression of interferon-regulated genes (IRGs) that might inhibit replication of the virus. Recently, we demonstrated that DBs induce a robust interferon response in the absence of infection. Little is known thus far, including how DBs influence HCMV infection and virus–host interaction. (2) Methods: Purified DBs were used to study the impact on virus replication and on the innate defense mechanisms of the cell. (3) Results: The incubation of cells with DBs at the time of infection had little effect on viral genome replication. Preincubation of DBs, however, led to a marked reduction in viral release from infected cells. These cells showed an enhancement of the cytopathic effect, associated with a moderate increase in early apoptosis. Despite virus-induced mechanisms to limit the interferon response, the induction of interferon-regulated genes (IRGs) was upregulated by DB treatment. (4) Conclusions: DBs sensitize cells against viral infection, comparable to the effects of interferons. The activities of these particles need to be considered when studying viral–host interaction.

Reactivation of latent cytomegalovirus (CMV) endangers the therapeutic success of hematopoietic cell transplantation (HCT) in tumor patients due to cytopathogenic virus spread that leads to organ manifestations of CMV disease, to interstitial pneumonia in particular. In cases of virus variants that are refractory to standard antiviral pharmacotherapy, immunotherapy by adoptive cell transfer (ACT) of virus-specific CD8 + T cells is the last resort to bridge the “protection gap” between hematoablative conditioning for HCT and endogenous reconstitution of antiviral immunity. We have used the well-established mouse model of CD8 + T-cell immunotherapy by ACT in a setting of experimental HCT and murine CMV (mCMV) infection to pursue the concept of improving the efficacy of ACT by therapeutic vaccination (TherVac) post-HCT. TherVac aims at restimulation and expansion of limited numbers of transferred antiviral CD8 + T cells within the recipient. Syngeneic HCT was performed with C57BL/6 mice as donors and recipients. Recipients were infected with recombinant mCMV (mCMV-SIINFEKL) that expresses antigenic peptide SIINFEKL presented to CD8 + T cells by the MHC class-I molecule K b . ACT was performed with transgenic OT-I CD8 + T cells expressing a T-cell receptor specific for SIINFEKL-K b . Recombinant human CMV dense bodies (DB-SIINFEKL), engineered to contain SIINFEKL within tegument protein pUL83/pp65, served for vaccination. DBs were chosen as they represent non-infectious, enveloped, and thus fusion-competent subviral particles capable of activating dendritic cells and delivering antigens directly into the cytosol for processing and presentation in the MHC class-I pathway. One set of our experiments documents the power of vaccination with DBs in protecting the immunocompetent host against a challenge infection. A further set of experiments revealed a significant improvement of antiviral control in HCT recipients by combining ACT with TherVac. In both settings, the benefit from vaccination with DBs proved to be strictly epitope-specific. The capacity to protect was lost when DBs included the peptide sequence SIINFEKA lacking immunogenicity and antigenicity due to C-terminal residue point mutation L8A, which prevents efficient proteasomal peptide processing and binding to K b . Our preclinical research data thus provide an argument for using pre-emptive TherVac to enhance antiviral protection by ACT in HCT recipients with diagnosed CMV reactivation.

(1) Background: Cells infected with the human cytomegalovirus (HCMV) produce subviral particles, termed dense bodies (DBs), both in-vitro and in-vivo. They are released from cells, comparable to infectious virions, and are enclosed by a membrane that resembles the viral envelope and mediates the entry into cells. To date, little is known about how the DB uptake influences the gene expression in target cells. The purpose of this study was to investigate the impact of DBs on cells, in the absence of a viral infection. (2) Methods: Mass spectrometry, immunoblot analyses, siRNA knockdown, and a CRISPR-CAS9 knockout, were used to investigate the changes in cellular gene expression following a DB exposure; (3) Results: A number of interferon-regulated genes (IRGs) were upregulated after the fibroblasts and endothelial cells were exposed to DBs. This upregulation was dependent on the DB entry and mediated by the type I interferon signaling through the JAK-STAT pathway. The induction of IRGs was mediated by the sensing of the DB-introduced DNA by the pattern recognition receptor cGAS. (4) Conclusions: The induction of a strong type I IFN response by DBs is a unique feature of the HCMV infection. The release of DBs may serve as a danger signal and concomitantly contribute to the induction of a strong, antiviral immune response.

Dense bodies (DB) are complex, noninfectious particles produced during CMVinfection containing envelope and tegument proteins that may be ideal candidates as vaccines. Although DB were previously described in fibroblasts, no evidence of DB formation has been shown after propagating CMV in epithelial cells. In the present study, both fibroblast MRC-5 and epithelial ARPE-19 cells were used to study DB production during CMV infection. We demonstrate the formation of epithelial cell-derived DB, mostly located as cytoplasmic inclusions in the perinuclear area of the infected cell. DB were gradient-purified, and the nature of the viral particles was confirmed using CMV-specific immunelabeling. Epithelial cell-derived DB had higher density and more homogeneous size (200–300 nm) compared to fibroblast-derived DB (100–600 nm).In agreement with previous results characterizing DB from CMV-infected fibroblasts, the pp65 tegument protein was predominant in the epithelial cell-derived DB. Our results also suggest that epithelial cells had more CMV capsids in the cytoplasm and had spherical bodies compatible with nucleus condensation (pyknosis) in cells undergoing apoptosis that were not detected in MRC-5 infected cells at the tested time post-infection. Our results demonstrate the formation of DB in CMV-infected ARPE-19 epithelial cells that may be suitable candidate to develop a multiprotein vaccine with antigenic properties similar to that of the virions while not including the viral genome.

Human cytomegalovirus (HCMV) infection is associated with severe disease conditions either following congenital transmission of the virus or viral reactivation in immunosuppressed individuals. Consequently, the establishment of a protective vaccine is of high medical need. Several candidates have been tested in preclinical and clinical studies, yet no vaccine has been licensed. Subviral dense bodies (DB) are a promising vaccine candidate. We have recently provided a GMP-compliant protocol for the production of DB, based on a genetically modified version of the HCMV laboratory strain Towne, expressing the pentameric complex of envelope protein gH-gL-pUL128-131 (Towne-UL130rep). In this work, we genetically attenuated Towne-UL130rep by abrogating the expression of the tegument protein pUL25 and by fusing the destabilizing domain ddFKBP to the N-terminus of the IE1- and IE2-proteins of HCMV. The resulting strain, termed TR-VAC, produced high amounts of DB under IE1/IE2 repressive conditions and concomitant supplementation of the viral terminase inhibitor letermovir to the producer cell culture. TR-VAC DB retained the capacity to induce neutralizing antibodies. A complex pattern of host protein induction was observed by mass spectrometry following exposure of primary human monocytes with TR-VAC DB. Human monocyte-derived dendritic cells (DC) moderately increased the expression of activation markers and MHC molecules upon stimulation with TR-VAC DB. In a co-culture with autologous T cells, the TR-VAC DB-stimulated DC induced a robust HCMV-specific T cell-activation and –proliferation. Exposure of donor-derived monocytic cells to DB led to the activation of a rapid innate immune response. This comprehensive data set thus shows that TR-VAC is an optimal attenuated seed virus strain for the production of a DB vaccine to be tested in clinical studies.

Congenital cytomegalovirus (cCMV) might occur as a result of the human cytomegalovirus (HCMV) primary (PI) or nonprimary infection (NPI) in pregnant women. Immune correlates of protection against cCMV have been partly identified only for PI. Following either PI or NPI, HCMV strains undergo latency. From a diagnostic standpoint, while the serological criteria for the diagnosis of PI are well-established, those for the diagnosis of NPI are still incomplete. Thus far, a recombinant gB subunit vaccine has provided the best results in terms of partial protection. This partial efficacy was hypothetically attributed to the post-fusion instead of the pre-fusion conformation of the gB present in the vaccine. Future efforts should be addressed to verify whether a new recombinant gB pre-fusion vaccine would provide better results in terms of prevention of both PI and NPI. It is still a matter of debate whether human hyperimmune globulin are able to protect from HCMV vertical transmission. In conclusion, the development of an HCMV vaccine that would prevent a significant portion of PI would be a major step forward in the development of a vaccine for both PI and NPI.

The morphogenesis of human cytomegalovirus (HCMV) particles is incompletely understood. Analysis of the protein composition of HCMV virions and subviral dense bodies (DBs) by mass spectrometry provides valuable information to increase our knowledge about viral morphogenesis. Here we addressed the viral proteome of virions and DBs from two fibroblast-passaged isolates and the widely used endotheliotropic TB4-BAC40 strain of HCMV. The results show a striking concordance of the particle proteomes of different strains. One surprising finding was that only low levels of gpUL128-131A were found in TB40-BAC4 virions. These three proteins, together with gH and gL, form a protein complex that is critical for the endothelial cell tropism of that strain. This indicates that either few molecules of that complex per virion or a small fraction of pentamer-positive virions suffice to retain the tropism. Furthermore, using a pp65-deficient variant of TB40-BAC4, we confirm our previous finding that the major tegument protein serves as a scaffold to support the upload of a fraction of the outer tegument proteins into particles. The results demonstrate that HCMV particle morphogenesis is an orchestrated process that leads to the formation of particles with a largely strain-independent protein composition.

Infections with the human cytomegalovirus (HCMV) are associated with severe clinical manifestations in children following prenatal transmission and after viral reactivation in immunosuppressed individuals. The development of an HCMV vaccine has long been requested but there is still no licensed product available. Subviral dense bodies (DB) are immunogenic in pre-clinical models and are thus a promising HCMV vaccine candidate. Recently, we established a virus based on the laboratory strain Towne that synthesizes large numbers of DB containing the pentameric protein complex gH/gL/UL128-131 (Towne-UL130repΔGFP). The work presented here focuses on providing strategies for the production of a safe vaccine based on that strain. A GMP-compliant protocol for DB production was established. Furthermore, the DB producer strain Towne-UL130rep was attenuated by deleting the UL25 open reading frame. Additional genetic modifications aim to abrogate its capacity to replicate in vivo by conditionally expressing pUL51 using the Shield-1/FKBP destabilization system. We further show that the terminase inhibitor letermovir can be used to reduce infectious virus contamination of a DB vaccine by more than two orders of magnitude. Taken together, strategies are provided here that allow for the production of a safe and immunogenic DB vaccine for clinical testing.

Cell-culture studies have revealed some of the fundamental features of the interaction of amyloid Abeta with cells and the mechanism of amyloid accumulation and pathogenesis in vitro. A(beta)1-42, the longer isoform of amyloid that is preferentially concentrated in senile plaque (SP) amyloid deposits in Alzheimer's disease (AD), is resistant to degradation and accumulates as insoluble aggregates in late endosomes or lysosomes. Once these aggregates have nucleated inside the cell, they grow by the addition of aberrantly folded APP and amyloidgenic fragments of APP, that would otherwise be degraded, onto the amyloid lattice in a fashion analogous to prion replication. This accumulation of heterogeneous aggregated APP fragments and Abeta appears to mimic the pathophysiologyof dystrophic neurites, where the same spectrum of components has been identified by immunohistochemistry. In the brain, this residue appears to be released into the extracellular space, possibly by a partially apoptotic mechanism that is restricted to the distal compartments of the neuron. Ultimately, this insoluble residue may be further digested to the protease-resistant A(beta)n-42 core, perhaps by microglia, where it accumulates as senile plaques. Thus, the dystrophic neurites are likely to be the source of the immediate precursors of amyloid in the senile plaques. This is the opposite of the commonly held view that extracellular accumulation of amyloid induces dystrophic neurites. Many of the key pathological events of AD may also be directly related to the intracellular accumulation of this insoluble amyloid. The aggregated, intracellular amyloid induces the production of reactive oxygen species (ROS) and lipid peroxidation products and ultimately results in the leakage of the lysosomal membrane. The breakdown of the lysosomal membrane may be a key pathogenic event, leading to the release of heparan sulfate and lysosomal hydrolases into the cytosol. Together, these observations provide the novel view that amyloid deposits and some of the early events of amyloid pathogenesis initiate randomly within single cells in AD. This pathogenic mechanism can explain some of the more enigmatic features of Alzheimer's pathogenesis, like the focal nature of amyloid plaques, the relationship between amyloid, dystrophic neurites and neurofibrillary-tangle pathology, and the miscompartmentalization of extracellular and cytosolic components observed in AD brain.

During mammalian meiosis, transcriptional silencing of the XY bivalent is a necessary event where defects may lead to infertility in males. While not well understood, the mechanism of meiotic gene silencing is believed to be RNA-dependent. In this study, we investigated the types and localization of non-coding RNAs in the meiotic nucleus of the male mouse using a microarray screen with different cell isolates as well as FISH. We report that the dense body, a component of the murine spermatocyte sex body similar to that of a dense body in Chinese hamster spermatocytes, is DNA-negative but rich in proteins and RNA including miRNAs (micro RNAs) and piRNAs (PIWI associated small RNAs), or their precursors. Selective miRNAs and piRNAs localize to chromosome cores, telomeres and the sex body of spermatocytes. These RNAs have not previously been detected in meiotic nuclei. These RNAs appear to associate with the nucleolus of the Sertoli cells as well as with the dense body. While in MIWI-null male mice the nucleolar signal from miRNA and piRNA probes in Sertoli cells is largely diminished, a differential regulation must exist in meiotic nuclei since the localization of these two components appears to be unaffected in the null animal.