Proteome changes of fibroblasts and endothelial cells upon incubation with human cytomegalovirus subviral Dense Bodies.

Human cytomegalovirus (HCMV) is a pathogen of high medical relevance. Subviral Dense Bodies (DB) were developed as a vaccine candidate to ameliorate the severe consequences of HCMV infection. Development of such a candidate vaccine for human application requires detailed knowledge of its interaction with the host. A comprehensive mass spectrometry (MS)- based analysis was performed regarding the changes in the proteome of cell culture cells, exposed to DB.

Subviral Dense Bodies of Human Cytomegalovirus Enhance Interferon-Beta Responses in Infected Cells and Impair Progeny Production.

(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.

Subviral Dense Bodies of Human Cytomegalovirus Induce an Antiviral Type I Interferon Response.

(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.

An Attenuated Strain of Human Cytomegalovirus for the Establishment of a Subviral Particle Vaccine.

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.

Production Strategies for Pentamer-Positive Subviral Dense Bodies as a Safe Human Cytomegalovirus Vaccine.

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.

Dense Bodies of a gH/gL/UL128/UL130/UL131 Pentamer-Repaired Towne Strain of Human Cytomegalovirus Induce an Enhanced Neutralizing Antibody Response.

The development of a vaccine against human cytomegalovirus infection (HCMV) is a high-priority medical goal. The viral pentameric protein complex consisting of glycoprotein H (gH)/gL/UL128-131A (PC) is considered to be an important vaccine component. Its relevance to the induction of a protective antibody response is, however, still a matter of debate. We addressed this issue by using subviral dense bodies (DBs) of HCMV. DBs are exceptionally immunogenic. Laboratory HCMV strain DBs harbor important neutralizing antibody targets, like the glycoproteins B, H, L, M, and N, but they are devoid of the PC. To be able to directly compare the impact of the PC on the levels of neutralizing antibody (NT-abs) responses, a PC-positive variant of the HCMV laboratory strain Towne was established by bacterial artificial chromosome (BAC) mutagenesis (Towne-UL130rep). This strain synthesized PC-positive DBs upon infection of fibroblasts. These DBs were used in side-by-side immunizations with PC-negative Towne DBs. Mouse and rabbit sera were tested to address the impact of the PC on DB immunogenicity. The neutralizing antibody response to PC-positive DBs was superior to that of PC-negative DBs, as tested on fibroblasts, epithelial cells, and endothelial cells and for both animal species used. The experiments revealed the potential of the PC to enhance the antibody response against HCMV. Of particular interest was the finding that PC-positive DBs induced an antibody response that blocked the infection of fibroblasts by a PC-positive viral strain more efficiently than sera following immunizations with PC-negative particles.IMPORTANCE Infections with the human cytomegalovirus (HCMV) may cause severe and even life-threatening disease manifestations in newborns and immunosuppressed individuals. Several strategies for the development of a vaccine against this virus are currently pursued. A critical question in this respect refers to the antigenic composition of a successful vaccine. Using a subviral particle vaccine candidate, we show here that one protein complex of HCMV, termed the pentameric complex (PC), enhances the neutralizing antibody response against viral infection of different cell types. We further show for the first time that this not only relates to the infection of epithelial or endothelial cells; the presence of the PC in the particles also enhanced the neutralizing antibody response against the infection of fibroblasts by HCMV. Together, these findings argue in favor of including the PC in strategies for HCMV vaccine development.

Targeting Nonsense Mutations in Diseases with Translational Read-Through-Inducing Drugs (TRIDs).

In recent years, remarkable advances in the ability to diagnose genetic disorders have been made. The identification of disease-causing genes allows the development of gene-specific therapies with the ultimate goal to develop personalized medicines for each patient according to their own specific genetic defect. In-depth genotyping of many different genes has revealed that ~12% of inherited genetic disorders are caused by in-frame nonsense mutations. Nonsense (non-coding) mutations are caused by point mutations, which generate premature termination codons (PTCs) that cause premature translational termination of the mRNA, and subsequently inhibit normal full-length protein expression. Recently, a gene-based therapeutic approach for genetic diseases caused by nonsense mutations has emerged, namely the so-called translational read-through (TR) therapy. Read-through therapy is based on the discovery that small molecules, known as TR-inducing drugs (TRIDs), allow the translation machinery to suppress a nonsense codon, elongate the nascent peptide chain, and consequently result in the synthesis of full-length protein. Several TRIDs are currently under investigation and research has been performed on several genetic disorders caused by nonsense mutations over the years. These findings have raised hope for the usage of TR therapy as a gene-based pharmacogenetic therapy for nonsense mutations in various genes responsible for a variety of genetic diseases.

Translational read-through as an alternative approach for ocular gene therapy of retinal dystrophies caused by in-frame nonsense mutations.

The eye has become an excellent target for gene therapy, and gene augmentation therapy of inherited retinal disorders has made major progress in recent years. Nevertheless, a recent study indicated that gene augmentation intervention might not stop the progression of retinal degeneration in patients. In addition, for many genes, viral-mediated gene augmentation is currently not feasible due to gene size and limited packaging capacity of viral vectors as well as expression of various heterogeneous isoforms of the target gene. Thus, alternative gene-based strategies to stop or delay the retinal degeneration are necessary. This review focuses on an alternative pharmacologic treatment strategy based on the usage of translational read-through inducing drugs (TRIDs) such as PTC124, aminoglycoside antibiotics, and designer aminoglycosides for overreading in-frame nonsense mutations. This strategy has emerged as an option for up to 30-50% of all cases of recessive hereditary retinal dystrophies. In-frame nonsense mutations are single-nucleotide alterations within the gene coding sequence resulting in a premature stop codon. Consequently, translation of such mutated genes leads to the synthesis of truncated proteins, which are unable to fulfill their physiologic functions. In this context, application of TRIDs facilitates the recoding of the premature termination codon into a sense codon, thus restoring syntheses of full-length proteins. So far, clinical trials for non-ocular diseases have been initiated for diverse TRIDs. Although the clinical outcome is not analyzed in detail, an excellent safety profile, namely for PTC124, was clearly demonstrated. Moreover, recent data demonstrated sustained read-through efficacies of nonsense mutations causing retinal degeneration, as manifested in the human Usher syndrome. In addition, a strong retinal biocompatibility for PTC124 and designer aminoglycosides has been demonstrated. In conclusion, recent progress emphasizes the potential of TRIDs as an alternative pharmacologic treatment strategy for treating nonsense mutation-based retinal disorders.