A HCMV pp65 polypeptide promotes the expansion of CD4+ and CD8+ T cells across a wide range of HLA specificities

Human Cytomegalovirus (HCMV) can cause life threatening disease in infected hosts. Immunization with HLA-restricted immunodominant synthetic peptides and adoptive transfer of epitope specific T cells have been envisaged to generate or boost HCMV specific cellular immunity, thereby preventing HCMV infection or reactivation. However, induction or expansion of T cells effective against HCMV are limited by the need of utilizing peptides with defined HLA restrictions. We took advantage of a combination of seven predictive algorithms to identify immunogenic peptides of potential use in the prevention or treatment of HCMV infection or reactivation. Here we describe a pp65 derived peptide (pp65(340-355), RQYDPVAALFFFDIDL: RQY16-mer), characterized by peculiar features. First, RQY-16mer is able to stimulate HCMV pp65 specific responses in both CD4+ and CD8+ T cells, restricted by a wide range of HLA class I and class II determinants. Second, RQY-16mer is able to induce an unusually wide range of effector functions in CD4+ T cells, including proliferation, killing of autologous HCMV infected target cells and cytokine production. Third, and most importantly, the RQY-16mer is able to stimulate CD4+ and CD8+ T cell responses in pharmacologically immunosuppressed patients. These data suggest that a single reagent might qualify as synthetic immunogen for potentially large populations exposed to HCMV infection or reactivation

Development of a High-Throughput Assay To Measure the Neutralization Capability of Anti-Cytomegalovirus Antibodies

Infection by human cytomegalovirus (CMV) elicits a strong humoral immune response and robust anti-CMV antibody production. Diagnosis of virus infection can be carried out by using a variety of serological assays; however, quantification of serum antibodies against CMV may not present an accurate measure of a patient's ability to control a virus infection. CMV strains that express green fluorescent protein (GFP) fusion proteins can be used as screening tools for evaluating characteristics of CMV infection in vitro. In this study, we employed a CMV virus strain, AD169, that ectopically expresses a yellow fluorescent protein (YFP) fused to the immediate-early 2 (IE2) protein product (AD169(IE2-YFP)) to quantify a CMV infection in human cells. We created a high-throughput cell-based assay that requires minimal amounts of material and provides a platform for rapid analysis of the initial phase of virus infection, including virus attachment, fusion, and immediate-early viral gene expression. The AD169(IE2-YFP) cell infection system was utilized to develop a neutralization assay with a monoclonal antibody against the viral surface glycoprotein gH. The high-throughput assay was extended to measure the neutralization capacity of serum from CMV-positive subjects. These findings describe a sensitive and specific assay for the quantification of a key immunological response that plays a role in limiting CMV dissemination and transmission. Collectively, we have demonstrated that a robust high-throughput infection assay can analyze the early steps of the CMV life cycle and quantify the potency of biological reagents to attenuate a virus infection

Human cytomegalovirus reprogrammes haematopoietic progenitor cells into immunosuppressive monocytes to achieve latency

The precise cell type hosting latent human cytomegalovirus (HCMV) remains elusive. Here, we report that HCMV reprogrammes human haematopoietic progenitor cells (HPCs) into a unique monocyte subset to achieve latency. Unlike conventional monocytes, this monocyte subset possesses higher levels of B7-H4, IL-10 and inducible nitric oxide synthase (iNOS), a longer lifespan and strong immunosuppressive capacity. Cell sorting of peripheral blood from latently infected human donors confirms that only this monocyte subset, representing less than 0.1% of peripheral mononuclear cells, is HCMV genome-positive but immediate-early-negative. Mechanistic studies demonstrate that HCMV promotes the differentiation of HPCs into this monocyte subset by activating cellular signal transducer and activator of transcription 3 (STAT3). In turn, this monocyte subset generates a high level of nitric oxide (NO) to silence HCMV immediate-early transcription and promote viral latency. By contrast, the US28-knockout HCMV mutant, which is incapable of activating STAT3, fails to reprogramme the HPCs and achieve latency. Our findings reveal that via activating the STAT3-iNOS-NO axis, HCMV differentiates human HPCs into a longevous, immunosuppressive monocyte subset for viral latency