An ELISPOT-Based Assay to Measure HBV-Specific CD8+ T Cell Responses in Immunocompetent Mice.

Despite some important limitations, immunocompetent mouse models of HBV replication remain an essential tool for studying cellular and humoral immunity to the virus. CD8+ T cells are a critical component of the immune response to HBV due to their ability to both kill virus-infected hepatocytes and produce cytokines such as IFN-γ that non-cytopathically inhibit virus replication. A number of techniques can be used to measure the magnitude, specificity, and functionality of HBV-specific CD8+ T cells, each having its own unique advantages. We describe here the enzyme-linked immunospot (ELISPOT)-based assay, which, compared to other methods, is sensitive, cost-effective, and rapid and requires relatively little optimization, specialized training, or equipment.

Virus-Like Vesicle-Based Therapeutic Vaccine Vectors for Chronic Hepatitis B Virus Infection.

UNLABELLED: More than 500,000 people die each year from the liver diseases that result from chronic hepatitis B virus (HBV) infection. Therapeutic vaccines, which aim to elicit an immune response capable of controlling the virus, offer a potential new treatment strategy for chronic hepatitis B. Recently, an evolved, high-titer vaccine platform consisting of Semliki Forest virus RNA replicons that express the vesicular stomatitis virus glycoprotein (VSV G) has been described. This platform generates virus-like vesicles (VLVs) that contain VSV G but no other viral structural proteins. We report here that the evolved VLV vector engineered to additionally express the HBV middle surface envelope glycoprotein (MHBs) induces functional CD8 T cell responses in mice. These responses were greater in magnitude and broader in specificity than those obtained with other immunization strategies, including recombinant protein and DNA. Additionally, a single immunization with VLV-MHBs protected mice from HBV hydrodynamic challenge, and this protection correlated with the elicitation of a CD8 T cell recall response. In contrast to MHBs, a VLV expressing HBV core protein (HBcAg) neither induced a CD8 T cell response in mice nor protected against challenge. Finally, combining DNA and VLV-MHBs immunization led to induction of HBV-specific CD8 T cell responses in a transgenic mouse model of chronic HBV infection. The ability of VLV-MHBs to induce a multispecific T cell response capable of controlling HBV replication, and to generate immune responses in a tolerogenic model of chronic infection, indicates that VLV vaccine platforms may offer a unique strategy for HBV therapeutic vaccination. IMPORTANCE: HBV infection is associated with significant morbidity and mortality. Furthermore, treatments for chronic infection are suboptimal and rarely result in complete elimination of the virus. Therapeutic vaccines represent a unique approach to HBV treatment and have the potential to induce long-term control of infection. Recently, a virus-based vector system that combines the nonstructural proteins of Semliki Forest virus with the VSV glycoprotein has been described. In this study, we used this system to construct a novel HBV vaccine and demonstrated that the vaccine is capable of inducing virus-specific immune responses in mouse models of acute and chronic HBV replication. These findings highlight the potential of this new vaccine system and support the idea that highly immunogenic vaccines, such as viral vectors, may be useful in the treatment of chronic hepatitis B.

Type III interferon attenuates a vesicular stomatitis virus-based vaccine vector.

UNLABELLED: Vesicular stomatitis virus (VSV) has been extensively studied as a vaccine vector and oncolytic agent. Nevertheless, safety concerns have limited its widespread use in humans. The type III lambda interferon (IFN-λ) family of cytokines shares common signaling pathways with the IFN-α/ß family and thus evokes similar antiviral activities. However, IFN-λ signals through a distinct receptor complex that is expressed in a cell type-specific manner, which restricts its activity to epithelial barriers, particularly those corresponding to the respiratory and gastrointestinal tracts. In this study, we determined how IFN-λ expression from recombinant VSV would influence vector replication, spread, and immunogenicity. We demonstrate that IFN-λ expression severely attenuates VSV in cell culture. In vivo, IFN-λ limits VSV replication in the mouse lung after intranasal administration and reduces virus spread to other organs. Despite this attenuation, however, the vector retains its capacity to induce protective CD8 T cell and antibody responses after a single immunization. These findings demonstrate a novel method of viral vector attenuation that could be used in both vaccine and oncolytic virus applications. IMPORTANCE: Viruses such as VSV that are used as vaccine vectors can induce protective T cell and antibody responses after a single dose. Additionally, IFN-λ is a potent antiviral agent that has certain advantages for clinical use compared to IFN-α/ß, such as fewer patient side effects. Here, we demonstrate that IFN-λ attenuates VSV replication and spread following intranasal virus delivery but does not reduce the ability of VSV to induce potent protective immune responses. These findings demonstrate that the type III IFN family may have widespread applicability for improving the safety and efficacy of viral vaccine and oncolytic vectors.

TRIM15 is a focal adhesion protein that regulates focal adhesion disassembly.

Focal adhesions are macromolecular complexes that connect the actin cytoskeleton to the extracellular matrix. Dynamic turnover of focal adhesions is crucial for cell migration. Paxillin is a multi-adaptor protein that plays an important role in regulating focal adhesion dynamics. Here, we identify TRIM15, a member of the tripartite motif protein family, as a paxillin-interacting factor and a component of focal adhesions. TRIM15 localizes to focal contacts in a myosin-II-independent manner by an interaction between its coiled-coil domain and the LD2 motif of paxillin. Unlike other focal adhesion proteins, TRIM15 is a stable focal adhesion component with restricted mobility due to its ability to form oligomers. TRIM15-depleted cells display impaired cell migration and reduced focal adhesion disassembly rates, in addition to enlarged focal adhesions. Thus, our studies demonstrate a cellular function for TRIM15 as a regulatory component of focal adhesion turnover and cell migration.

Functional characterization of the putative hepatitis B virus core protein late domain using retrovirus chimeras.

The hepatitis B virus (HBV) Core protein encodes a late (L)-domain like motif (129PPAYRPPNAP(138)) that has been purported to serve as a docking site for recruitment of host factors such as Nedd4 that can mediate viral particle release from infected cells. However, mutation of this region of Core typically disrupts nucleocapsid formation in the cytoplasm, making it difficult to ascertain if the Core PPAY motif constitutes a functional L-domain that mediates HBV release in the context of replicating virus. Since many viral L-domains are functionally interchangeable between different virus families, and such swapping experiments have been used as a tool to identify other viral sequences with L-domain activity, we generated chimeric constructs between murine leukemia virus (MLV) Gag and HBV Core to determine if the potential HBV L-domain motif is sufficient to stimulate virus release. We found that the HBV Core PPAY motif, but not the PNAP motif, demonstrates L-domain activity in the context of MLV replication to direct virus release and infectious virion production. Additionally, we found that overexpression of the cellular Nedd4 or WWP1 ubiquitin ligases stimulates release of a partially defective PPAY domain mutant, providing further evidence supporting a role for the Nedd4 ubiquitin ligase in promoting HBV release. These studies lend further insight into the mechanisms used by HBV to mediate its release from infected cells.