Atypical B cells and impaired SARS-CoV-2 neutralization following heterologous vaccination in the elderly.

Suboptimal responses to a primary vaccination course have been reported in the elderly, but there is little information regarding the impact of age on responses to booster third doses. Here, we show that individuals 70 years or older (median age 73, range 70-75) who received a primary two-dose schedule with AZD1222 and booster third dose with mRNA vaccine achieve significantly lower neutralizing antibody responses against SARS-CoV-2 spike pseudotyped virus compared with those younger than 70 (median age 66, range 54-69) at 1 month post booster. Impaired neutralization potency and breadth post third dose in the elderly is associated with circulating "atypical" spike-specific B cells expressing CD11c and FCRL5. However, when considering individuals who received three doses of mRNA vaccine, we did not observe differences in neutralization or enrichment in atypical B cells. This work highlights the finding that AdV and mRNA COVID-19 vaccine formats differentially instruct the memory B cell response.

Differential dysregulation of ß-TrCP1 and -2 by HIV-1 Vpu leads to inhibition of canonical and non-canonical NF-κB pathways in infected cells.

The HIV-1 Vpu protein is expressed late in the virus lifecycle to promote infectious virus production and avoid innate and adaptive immunity. This includes the inhibition of the NF-κB pathway which, when activated, leads to the induction of inflammatory responses and the promotion of antiviral immunity. Here we demonstrate that Vpu can inhibit both canonical and non-canonical NF-κB pathways, through the direct inhibition of the F-box protein ß-TrCP, the substrate recognition portion of the Skp1-Cul1-F-box (SCF)ß-TrCP ubiquitin ligase complex. There are two paralogues of ß-TrCP (ß-TrCP1/BTRC and ß-TrCP2/FBXW11), encoded on different chromosomes, which appear to be functionally redundant. Vpu, however, is one of the few ß-TrCP substrates to differentiate between the two paralogues. We have found that patient-derived alleles of Vpu, unlike those from lab-adapted viruses, trigger the degradation of ß-TrCP1 while co-opting its paralogue ß-TrCP2 for the degradation of cellular targets of Vpu, such as CD4. The potency of this dual inhibition correlates with stabilization of the classical IκBα and the phosphorylated precursors of the mature DNA-binding subunits of canonical and non-canonical NF-κB pathways, p105/NFκB1 and p100/NFκB2, in HIV-1 infected CD4+ T cells. Both precursors act as alternative IκBs in their own right, thus reinforcing NF-κB inhibition at steady state and upon activation with either selective canonical or non-canonical NF-κB stimuli. These data reveal the complex regulation of NF-κB late in the viral replication cycle, with consequences for both the pathogenesis of HIV/AIDS and the use of NF-κB-modulating drugs in HIV cure strategies. IMPORTANCE The NF-κB pathway regulates host responses to infection and is a common target of viral antagonism. The HIV-1 Vpu protein inhibits NF-κB signaling late in the virus lifecycle, by binding and inhibiting ß-TrCP, the substrate recognition portion of the ubiquitin ligase responsible for inducing IκB degradation. Here we demonstrate that Vpu simultaneously inhibits and exploits the two different paralogues of ß-TrCP by triggering the degradation of ß-TrCP1 and co-opting ß-TrCP2 for the destruction of its cellular targets. In so doing, it has a potent inhibitory effect on both the canonical and non-canonical NF-κB pathways. This effect has been underestimated in previous mechanistic studies due to the use of Vpu proteins from lab-adapted viruses. Our findings reveal previously unappreciated differences in the ß-TrCP paralogues, revealing functional insights into the regulation of these proteins. This study also raises important implications for the role of NF-κB inhibition in the immunopathogenesis of HIV/AIDS and the way that this may impact on HIV latency reversal strategies based on the activation of the non-canonical NF-κB pathway.

Human Cytomegalovirus Infection of Epithelial Cells Increases SARS-CoV-2 Superinfection by Upregulating the ACE2 Receptor.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has caused widespread morbidity and mortality since its onset in late 2019. Here, we demonstrate that prior infection with human cytomegalovirus (HCMV) substantially increases infection with SARS-CoV-2 in vitro. HCMV is a common herpesvirus carried by 40%-100% of the population, which can reactivate in the lung under inflammatory conditions, such as those resulting from SARS-CoV-2 infection. We show in both endothelial and epithelial cell types that HCMV infection upregulates ACE2, the SARS-CoV-2 cell entry receptor. These observations suggest that HCMV reactivation events in the lung of healthy HCMV carriers could exacerbate SARS-CoV-2 infection and subsequent COVID-19 symptoms. This effect could contribute to the disparity of disease severity seen in ethnic minorities and those with lower socioeconomic status, due to their higher CMV seroprevalence. Our results warrant further clinical investigation as to whether HCMV infection influences the pathogenesis of SARS-CoV-2.

The Human Cytomegalovirus ß2.7 Long Non-Coding RNA Prevents Induction of Reactive Oxygen Species to Maintain Viral Gene Silencing during Latency.

Human cytomegalovirus (HCMV) is a significant source of disease for the immunosuppressed and immunonaive. The treatment of HCMV is made more problematic by viral latency, a lifecycle stage in which the virus reduces its own gene expression and produces no infectious virus. The most highly expressed viral gene during HCMV latency is the viral ß2.7 long non-coding RNA. Although we have recently shown that the ß2.7 lncRNA lowers levels of reactive oxygen species (ROS) during infection in monocytes, how this impacts latency is unclear. We now show that ß2.7 is important for establishing and maintaining HCMV latency by aiding the suppression of viral lytic gene expression and that this is directly related to its ability to quench reactive oxygen species (ROS). Consistent with this, we also find that exogenous inducers of ROS cause reactivation of latent HCMV. These effects can be compensated by treatment with an antioxidant to lower ROS levels. Finally, we show that ROS-mediated reactivation is independent of myeloid differentiation, but instead relies on NF-κB activation. Altogether, these results reveal a novel factor that is central to the complex process that underpins HCMV latency. These findings may be of particular relevance in the transplant setting, in which transplanted tissue/organs are subject to very high ROS levels, and HCMV reactivation poses a significant threat.

A Viral Long Non-Coding RNA Protects against Cell Death during Human Cytomegalovirus Infection of CD14+ Monocytes.

Long non-coding RNA ß2.7 is the most highly transcribed viral gene during latent human cytomegalovirus (HCMV) infection. However, as yet, no function has ever been ascribed to ß2.7 during HCMV latency. Here we show that ß2.7 protects against apoptosis induced by high levels of reactive oxygen species (ROS) in infected monocytes, which routinely support latent HCMV infection. Monocytes infected with a wild-type (WT) virus, but not virus deleted for the ß2.7 gene (Δß2.7), are protected against mitochondrial stress and subsequent apoptosis. Protected monocytes display lower levels of ROS and additionally, stress-induced death in the absence of ß2.7 can be reversed by an antioxidant which reduces ROS levels. Furthermore, we show that infection with WT but not Δß2.7 virus results in strong upregulation of a cellular antioxidant enzyme, superoxide dismutase 2 (SOD2) in CD14+ monocytes. These observations identify a role for the ß2.7 viral transcript, the most abundantly expressed viral RNA during latency but for which no latency-associated function has ever been ascribed, and demonstrate a novel way in which HCMV protects infected monocytes from pro-death signals to optimise latent carriage.

HCMV Antivirals and Strategies to Target the Latent Reservoir.

Human cytomegalovirus (HCMV) is a ubiquitous human herpesvirus. In healthy people, primary infection is generally asymptomatic, and the virus can go on to establish lifelong latency in cells of the myeloid lineage. However, HCMV often causes severe disease in the immunosuppressed: transplant recipients and people living with AIDS, and also in the immunonaive foetus. At present, there are several antiviral drugs licensed to control HCMV disease. However, these are all faced with problems of poor bioavailability, toxicity and rapidly emerging viral resistance. Furthermore, none of them are capable of fully clearing the virus from the host, as they do not target latent infection. Consequently, reactivation from latency is a significant source of disease, and there remains an unmet need for treatments that also target latent infection. This review briefly summarises the most common HCMV antivirals used in clinic at present and discusses current research into targeting the latent HCMV reservoir.

A BMPR2/YY1 Signaling Axis Is Required for Human Cytomegalovirus Latency in Undifferentiated Myeloid Cells.

Human cytomegalovirus (HCMV) presents a major health burden in the immunocompromised and in stem cell transplant medicine. A lack of understanding about the mechanisms of HCMV latency in undifferentiated CD34+ stem cells, and how latency is broken for the virus to enter the lytic phase of its infective cycle, has hampered the development of essential therapeutics. Using a human induced pluripotent stem cell (iPSC) model of HCMV latency and patient-derived myeloid cell progenitors, we demonstrate that bone morphogenetic protein receptor type 2 (BMPR2) is necessary for HCMV latency. In addition, we define a crucial role for the transcription factor Yin Yang 1 (YY1) in HCMV latency; high levels of YY1 are maintained in latently infected cells as a result of BMPR2 signaling through the SMAD4/SMAD6 axis. Activation of SMAD4/6, through BMPR2, inhibits TGFbeta receptor signaling, which leads to the degradation of YY1 via induction of a cellular microRNA (miRNA), hsa-miR-29a. Pharmacological targeting of BMPR2 in progenitor cells results in the degradation of YY1 and an inability to maintain latency and renders cells susceptible to T cell killing. These data argue that BMPR2 plays a role in HCMV latency and is a new potential therapeutic target for maintaining or disrupting HCMV latency in myeloid progenitors. IMPORTANCE Understanding the mechanisms which regulate HCMV latency could allow therapeutic targeting of the latent virus reservoir from where virus reactivation can cause severe disease. We show that the BMPR2/TGFbeta receptor/YY1 signaling axis is crucial to maintain HCMV latency in undifferentiated cells and that pharmacological reduction of BMPR2 in latently infected cells leads to reactivation of the viral lytic transcription program, which renders the infected cell open to immune detection and clearance in infected individuals. Therefore, this work identifies key host-virus interactions which regulate HCMV latent infection. It also demonstrates a potential new therapeutic approach to reduce HCMV reactivation-mediated disease by the treatment of donor stem cells/organs prior to transplantation, which could have a major impact in the transplant disease setting.

Regulation of host and viral promoters during human cytomegalovirus latency via US28 and CTCF.

Viral latency is an active process during which the host cell environment is optimized for latent carriage and reactivation. This requires control of both viral and host gene promoters and enhancers often at the level of chromatin, and several viruses co-opt the chromatin organiser CTCF to control gene expression during latency. While CTCF has a role in the latencies of alpha- and gamma-herpesviruses, it was not known whether CTCF played a role in the latency of the beta-herpesvirus human cytomegalovirus (HCMV). Here, we show that HCMV latency is associated with increased CTCF expression and CTCF binding to the viral major lytic promoter, the major immediate early promoter (MIEP). This increase in CTCF binding is dependent on the virally encoded G protein coupled receptor, US28, and contributes to suppression of MIEP-driven transcription, a hallmark of latency. Furthermore, we show that latency-associated upregulation of CTCF represses expression of the neutrophil chemoattractants S100A8 and S100A9 which we have previously shown are downregulated during HCMV latency. As with downregulation of the MIEP, CTCF binding to the enhancer region of S100A8/A9 drives their suppression, again in a US28-dependent manner. Taken together, we identify CTCF upregulation as an important mechanism for optimizing latent carriage of HCMV at both the levels of viral and cellular gene expression.