Cytomegalovirus Vaccine Strain Towne-Derived Dense Bodies Induce Broad Cellular Immune Responses and Neutralizing Antibodies That Prevent Infection of Fibroblasts and Epithelial Cells

Human cytomegalovirus (HCMV), a betaherpesvirus, can cause severe disease in immunosuppressed patients and following congenital infection. A vaccine that induces both humoral and cellular immunity may be required to prevent congenital infection. Dense bodies (DBs) are complex, noninfectious particles produced by HCMV-infected cells and may represent a vaccine option. As knowledge of the antigenicity and immunogenicity of DB is incomplete, we explored characterization methods and defined DB production methods, followed by systematic evaluation of neutralization and cell-mediated immune responses to the DB material in BALB/c mice. DBs purified from Towne-infected cultures treated with the viral terminase inhibitor 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB) were characterized by nanoparticle tracking analysis (NTA), two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), immunoblotting, quantitative enzyme-linked immunosorbent assay, and other methods. The humoral and cellular immune responses to DBs were compared to the immunogenicity of glycoprotein B (gB) administered with the adjuvant AddaVax (gB/AddaVax). DBs induced neutralizing antibodies that prevented viral infection of cultured fibroblasts and epithelial cells and robust cell-mediated immune responses to multiple viral proteins, including pp65, gB, and UL48. In contrast, gB/AddaVax failed to induce neutralizing antibodies that prevented infection of epithelial cells, highlighting a critical difference in the humoral responses induced by these vaccine candidates. Our data advance the potential for the DB vaccine approach, demonstrate important immunogenicity properties, and strongly support the further evaluation of DBs as a CMV vaccine candidate.     

S-glutathionylation activates STIM1 and alters mitochondrial homeostasis

Oxidant stress influences many cellular processes, including cell growth, differentiation, and cell death. A well-recognized link between these processes and oxidant stress is via alterations in Ca²⁺ signaling. However, precisely how oxidants influence Ca²⁺ signaling remains unclear. Oxidant stress led to a phenotypic shift in Ca²⁺ mobilization from an oscillatory to a sustained elevated pattern via calcium release–activated calcium (CRAC)–mediated capacitive Ca²⁺ entry, and stromal interaction molecule 1 (STIM1)– and Orai1-deficient cells are resistant to oxidant stress. Functionally, oxidant-induced Ca²⁺ entry alters mitochondrial Ca²⁺ handling and bioenergetics and triggers cell death. STIM1 is S-glutathionylated at cysteine 56 in response to oxidant stress and evokes constitutive Ca²⁺ entry independent of intracellular Ca²⁺ stores. These experiments reveal that cysteine 56 is a sensor for oxidant-dependent activation of STIM1 and demonstrate a molecular link between oxidant stress and Ca²⁺ signaling via the CRAC channel.     

Histone Deacetylase Inhibitor Romidepsin Induces HIV Expression in CD4 T Cells from Patients on Suppressive Antiretroviral Therapy at Concentrations Achieved by Clinical Dosing

Persistent latent reservoir of replication-competent proviruses in memory CD4 T cells is a major obstacle to curing HIV infection. Pharmacological activation of HIV expression in latently infected cells is being explored as one of the strategies to deplete the latent HIV reservoir. In this study, we characterized the ability of romidepsin (RMD), a histone deacetylase inhibitor approved for the treatment of T-cell lymphomas, to activate the expression of latent HIV. In an in vitro T-cell model of HIV latency, RMD was the most potent inducer of HIV (EC₅₀ = 4.5 nM) compared with vorinostat (VOR; EC₅₀ = 3,950 nM) and other histone deacetylase (HDAC) inhibitors in clinical development including panobinostat (PNB; EC₅₀ = 10 nM). The HIV induction potencies of RMD, VOR, and PNB paralleled their inhibitory activities against multiple human HDAC isoenzymes. In both resting and memory CD4 T cells isolated from HIV-infected patients on suppressive combination antiretroviral therapy (cART), a 4-hour exposure to 40 nM RMD induced a mean 6-fold increase in intracellular HIV RNA levels, whereas a 24-hour treatment with 1 µM VOR resulted in 2- to 3-fold increases. RMD-induced intracellular HIV RNA expression persisted for 48 hours and correlated with sustained inhibition of cell-associated HDAC activity. By comparison, the induction of HIV RNA by VOR and PNB was transient and diminished after 24 hours. RMD also increased levels of extracellular HIV RNA and virions from both memory and resting CD4 T-cell cultures. The activation of HIV expression was observed at RMD concentrations below the drug plasma levels achieved by doses used in patients treated for T-cell lymphomas. In conclusion, RMD induces HIV expression ex vivo at concentrations that can be achieved clinically, indicating that the drug may reactivate latent HIV in patients on suppressive cART.     

Requirement of FADD, NEMO, and BAX/BAK for aberrant mitochondrial function in tumor necrosis factor alpha-induced necrosis

Necroptosis represents a form of alternative programmed cell death that is dependent on the kinase RIP1. RIP1-dependent necroptotic death manifests as increased reactive oxygen species (ROS) production in mitochondria and is accompanied by loss of ATP biogenesis and eventual dissipation of mitochondrial membrane potential. Here, we show that tumor necrosis factor alpha (TNF-α)-induced necroptosis requires the adaptor proteins FADD and NEMO. FADD was found to mediate formation of the TNF-α-induced pronecrotic RIP1-RIP3 kinase complex, whereas the IκB Kinase (IKK) subunit NEMO appears to function downstream of RIP1-RIP3. Interestingly, loss of RelA potentiated TNF-α-dependent necroptosis, indicating that NEMO regulates necroptosis independently of NF-κB. Using both pharmacologic and genetic approaches, we demonstrate that the overexpression of antioxidants alleviates ROS elevation and necroptosis. Finally, elimination of BAX and BAK or overexpression of Bcl-x(L) protects cells from necroptosis at a later step. These findings provide evidence that mitochondria play an amplifying role in inflammation-induced necroptosis.     

Nitration of the mitochondrial complex I subunit NDUFB8 elicits RIP1- and RIP3-mediated necrosis

Nitric oxide (NO) and other reactive nitrogen species target multiple sites in the mitochondria to influence cellular bioenergetics and survival. Kinetic imaging studies revealed that NO from either activated macrophages or donor compounds rapidly diffuses to the mitochondria, causing a dose-dependent progressive increase in NO-dependent DAF fluorescence, which corresponded to mitochondrial membrane potential loss and initiated alterations in cellular bioenergetics that ultimately led to necrotic cell death. Cellular dysfunction is mediated by an elevated 3-nitrotyrosine signature of the mitochondrial complex I subunit NDUFB8, which is vital for normal mitochondrial function as evidenced by selective knockdown via siRNA. Overexpression of mitochondrial superoxide dismutase substantially decreased NDUFB8 nitration and restored mitochondrial homeostasis. Further, treatment of cells with either necrostatin-1 or siRNA knockdown of RIP1 and RIP3 prevented NO-mediated necrosis. This work demonstrates that the interaction between NO and mitochondrially derived superoxide alters mitochondrial bioenergetics and cell function, thus providing a molecular mechanism for reactive oxygen and nitrogen species-mediated alterations in mitochondrial homeostasis.