The Miami Monkey: A Sunny Alternative to the Berlin Patient.

Curing HIV infection has been impossible, with the exception of the "Berlin Patient." Martinez-Navio et al. (2019) in Miami herein present a rare monkey whose virus was controlled for >3 years after a single genetic intervention that led to persistent production of HIV-neutralizing antibodies in vivo.

Inhibition of spumavirus gene expression by PHF11.

The foamy viruses (FV) or spumaviruses are an ancient subfamily of retroviruses that infect a variety of vertebrates. FVs are endemic, but apparently apathogenic, in modern non-human primates. Like other retroviruses, FV replication is inhibited by type-I interferon (IFN). In a previously described screen of IFN-stimulated genes (ISGs), we identified the macaque PHD finger domain protein-11 (PHF11) as an inhibitor of prototype foamy virus (PFV) replication. Here, we show that human and macaque PHF11 inhibit the replication of multiple spumaviruses, but are inactive against several orthoretroviruses. Analysis of other mammalian PHF11 proteins revealed that antiviral activity is host species dependent. Using multiple reporter viruses and cell lines, we determined that PHF11 specifically inhibits a step in the replication cycle that is unique to FVs, namely basal transcription from the FV internal promoter (IP). In so doing, PHF11 prevents expression of the viral transactivator Tas and subsequent activation of the viral LTR promoter. These studies reveal a previously unreported inhibitory mechanism in mammalian cells, that targets a family of ancient viruses and may promote viral latency.

Sensing retroviruses.

Sensing pathogens is an essential first step in the initiation of a host response to infection. In this issue of Immunity, Kane et al. (2011) used mouse models to demonstrate that Toll-like receptor 7 is required for the generation of an antibody response to infection by retroviruses.

Tetherin Inhibits Cell-Free Virus Dissemination and Retards Murine Leukemia Virus Pathogenesis.

The relative contributions of cell-free virion circulation and direct cell-to-cell transmission to retroviral dissemination and pathogenesis are unknown. Tetherin/Bst2 is an antiviral protein that blocks enveloped virion release into the extracellular milieu but may not inhibit cell-to-cell virus transmission. We developed live-cell imaging assays which show that tetherin does not affect Moloney murine leukemia virus (MoMLV) spread, and only minimally affects vesicular stomatitis virus (VSV) spread, to adjacent cells in a monolayer. Conversely, cell-free MLV and VSV virion yields and VSV spread to distal cells were dramatically reduced by tetherin. To elucidate the roles of tetherin and cell-free virions during in vivo viral dissemination and pathogenesis, we developed mice carrying an inducible human tetherin (hTetherin) transgene. While ubiquitous hTetherin expression was detrimental to the growth and survival of mice, restriction of hTetherin expression to hematopoietic cells gave apparently healthy mice. The expression of hTetherin in hematopoietic cells had little or no effect on the number of MoMLV-infected splenocytes and thymocytes. However, hTetherin expression significantly reduced cell-free plasma viremia and also delayed MoMLV-induced disease. Overall, these results suggest that MoMLV spread within hematopoietic tissues and cell monolayers involves cell-to-cell transmission that is resistant to tetherin but that virion dissemination via plasma is inhibited by tetherin and is required for full MoMLV pathogenesis.IMPORTANCE Retroviruses are thought to spread primarily via direct cell-to-cell transmission, yet many have evolved to counteract an antiviral protein called tetherin, which may selectively inhibit cell-free virus release. We generated a mouse model with an inducible tetherin transgene in order to study how tetherin affects retroviral dissemination and on which cell types its expression is required to do so. We first developed a novel in vitro live-cell imaging assay to demonstrate that while tetherin does indeed dramatically reduce cell-free virus spreading, it has little to no effect on direct cell-to-cell transmission of either vesicular stomatitis virus (VSV) or the retrovirus MoMLV. Using our transgenic mouse model, we found that tetherin expression on hematopoietic cells resulted in the specific reduction of MoMLV cell-free plasma viremia but not the number of infected hematopoietic cells. The delay in disease associated with this scenario suggests a role for cell-free virus in retroviral disease progression.

SAMHD1-dependent retroviral control and escape in mice.

SAMHD1 is a host restriction factor for human immunodeficiency virus 1 (HIV-1) in cultured human cells. SAMHD1 mutations cause autoimmune Aicardi-Goutières syndrome and are found in cancers including chronic lymphocytic leukaemia. SAMHD1 is a triphosphohydrolase that depletes the cellular pool of deoxynucleoside triphosphates, thereby preventing reverse transcription of retroviral genomes. However, in vivo evidence for SAMHD1's antiviral activity has been lacking. We generated Samhd1 null mice that do not develop autoimmune disease despite displaying a type I interferon signature in spleen, macrophages and fibroblasts. Samhd1(-/-) cells have elevated deoxynucleoside triphosphate (dNTP) levels but, surprisingly, SAMHD1 deficiency did not lead to increased infection with VSV-G-pseudotyped HIV-1 vectors. The lack of restriction is likely attributable to the fact that dNTP concentrations in SAMHD1-sufficient mouse cells are higher than the KM of HIV-1 reverse transcriptase (RT). Consistent with this notion, an HIV-1 vector mutant bearing an RT with lower affinity for dNTPs was sensitive to SAMHD1-dependent restriction in cultured cells and in mice. This shows that SAMHD1 can restrict lentiviruses in vivo and that nucleotide starvation is an evolutionarily conserved antiviral mechanism.

Tetherin promotes the innate and adaptive cell-mediated immune response against retrovirus infection in vivo.

Tetherin/BST-2 is a host restriction factor that could directly inhibit retroviral particle release by tethering nascent virions to the plasma membrane. However, the immunological impact of Tetherin during retrovirus infection remains unknown. We now show that Tetherin influences antiretroviral cell-mediated immune responses. In contrast to the direct antiviral effects of Tetherin, which are dependent on cell surface expression, the immunomodulatory effects are linked to the endocytosis of the molecule. Mice encoding endocytosis-competent C57BL/6 Tetherin exhibited lower viremia and pathology at 7 d postinfection with Friend retrovirus (FV) compared with mice encoding endocytosis-defective NZW/LacJ Tetherin. Notably, antiretroviral protection correlated with stronger NK cell responses. In addition, Friend retrovirus infection levels were significantly lower in wild-type C57BL/6 mice than in Tetherin knockout mice at 2 wk postinfection, and antiretroviral protection correlated with stronger NK cell and virus-specific CD8+ T cell responses. The results demonstrate that Tetherin acts as a modulator of the cell-mediated immune response against retrovirus infection in vivo.

Tetherin is a key effector of the antiretroviral activity of type I interferon in vitro and in vivo.

Tetherin (Bst-2 CD317) is a cell-surface protein whose expression is induced by IFNα. Although tetherin expression causes the retention of retrovirus particles on the surface of infected cells, it is not known whether tetherin inhibits retroviral replication or pathogenesis in vivo. Mutation of tetherin antagonists often has little effect on retroviral replication in vitro, and, although tetherin can reduce the yield of extracellular viral particles, some studies suggest that tetherin actually enhances direct cell-to-cell viral transmission. We generated tetherin-deficient mice to determine the effect of this protein on murine retrovirus replication and pathogenesis. We find that tetherin markedly inhibits the replication of Moloney murine leukemia virus (Mo-MLV) and is required for the antiretroviral activity of IFNα to be fully manifested in vitro. Surprisingly, Mo-MLV replication and disease progression was not significantly different in WT and tetherin-deficient mice, but this finding was explained by the fact that Mo-MLV infection did not induce detectable tetherin expression on candidate target cells in vivo. Indeed, IFNα induction was required to reveal the anti-Mo-MLV activity of tetherin in vivo. Moreover, LP-BM5, an MLV strain that has been demonstrated to induce immune activation and IFNα expression, achieved higher levels of viremia and induced exaggerated pathology in tetherin-deficient mice. These data indicate that tetherin is a bona fide antiviral protein and can reduce retroviral replication and disease in vivo.

Delivery of DNA-Based Therapeutics for Treatment of Chronic Diseases.

Gene therapy and its role in the medical field have evolved drastically in recent decades. Studies aim to define DNA-based medicine as well as encourage innovation and the further development of novel approaches. Gene therapy has been established as an alternative approach to treat a variety of diseases. Its range of mechanistic applicability is wide; gene therapy has the capacity to address the symptoms of disease, the body's ability to fight disease, and in some cases has the ability to cure disease, making it a more attractive intervention than some traditional approaches to treatment (i.e., medicine and surgery). Such versatility also suggests gene therapy has the potential to address a greater number of indications than conventional treatments. Many DNA-based therapies have shown promise in clinical trials, and several have been approved for use in humans. Whereas current treatment regimens for chronic disease often require frequent dosing, DNA-based therapies can produce robust and durable expression of therapeutic genes with fewer treatments. This benefit encourages the application of DNA-based gene therapy to manage chronic diseases, an area where improving efficiency of current treatments is urgent. Here, we provide an overview of two DNA-based gene therapies as well as their delivery methods: adeno associated virus (AAV)-based gene therapy and plasmid DNA (pDNA)-based gene therapy. We will focus on how these therapies have already been utilized to improve treatment of chronic disease, as well as how current literature supports the expansion of these therapies to treat additional chronic indications in the future.

Enhancing In Vivo Electroporation Efficiency through Hyaluronidase: Insights into Plasmid Distribution and Optimization Strategies.

Electroporation (EP) stands out as a promising non-viral plasmid delivery strategy, although achieving optimal transfection efficiency in vivo remains a challenge. A noteworthy advancement in the field of in vivo EP is the application of hyaluronidase, an enzyme with the capacity to degrade hyaluronic acid in the extracellular matrix, which thereby enhances DNA transfer efficiency by 2- to 3-fold. This paper focuses on elucidating the mechanism of hyaluronidase's impact on transfection efficiency. We demonstrate that hyaluronidase promotes a more uniform distribution of plasmid DNA (pDNA) within skeletal muscle. Additionally, our study investigates the effect of the timing of hyaluronidase pretreatment on EP efficiency by including time intervals of 0, 5, and 30 min between hyaluronidase treatment and the application of pulses. Serum levels of the pDNA-encoded transgene reveal a minimal influence of the hyaluronidase pretreatment time on the final serum protein levels following delivery in both mice and rabbit models. Leveraging bioimpedance measurements, we capture morphological changes in muscle induced by hyaluronidase treatment, which result in a varied pDNA distribution. Subsequently, these findings are employed to optimize EP electrical parameters following hyaluronidase treatment in animal models. This paper offers novel insights into the potential of hyaluronidase in enhancing the effectiveness of in vivo EP, as well as guides optimized electroporation strategies following hyaluronidase use.

NF-kappaB activity is constitutively elevated in c-Abl null fibroblasts.

The c-abl proto-oncogene encodes a nonreceptor tyrosine kinase involved in many cellular processes, including signaling from growth factor and antigen receptors, remodeling the cytoskeleton, and responding to DNA damage and oxidative stress. Many downstream pathways are affected by c-Abl. Elevated c-Abl kinase activity can inhibit NF-kappaB activity by stabilizing the inhibitory protein IkappaB alpha, raising the possibility that c-Abl-deficient cells might have increased NF-kappaB activity. We examined the levels of NF-kappaB activity in primary mouse embryonic fibroblasts (MEFs) derived from wild-type and c-Abl knockout mice and found that the knockout MEFs indeed exhibited elevated NF-kappaB activity in response to stimulation as well as constitutively elevated NF-kappaB activity. Thus, endogenous c-Abl is a negative regulator of basal and inducible NF-kappaB activity. Examination of various points of NF-kappaB regulation revealed that unstimulated c-Abl knockout MEFs do not exhibit an increase in IkappaB alpha degradation, p65/RelA nuclear translocation, or DNA binding of NF-kappaB subunits. They do, however, show reduced levels of the histone deacetylase HDAC1, a negative regulator of basal NF-kappaB activity. Unstimulated c-Abl knockout MEFs are less responsive to induction of NF-kappaB activity by trichostatin A, an HDAC inhibitor, suggesting that c-Abl might play a role in the HDAC-mediated repression of basal NF-kappaB activity.

In Vivo Electroporation of Plasmid DNA: A Promising Strategy for Rapid, Inexpensive, and Flexible Delivery of Anti-Viral Monoclonal Antibodies.

Since the first approval of monoclonal antibodies by the United States Food and Drug Administration (FDA) in 1986, therapeutic antibodies have become one of the predominant classes of drugs in oncology and immunology. Despite their natural function in contributing to antiviral immunity, antibodies as drugs have only more recently been thought of as tools for combating infectious diseases. Passive immunization, or the delivery of the products of an immune response, offers near-immediate protection, unlike the active immune processes triggered by traditional vaccines, which rely on the time it takes for the host's immune system to develop an effective defense. This rapid onset of protection is particularly well suited to containing outbreaks of emerging viral diseases. Despite these positive attributes, the high cost associated with antibody manufacture and the need for a cold chain for storage and transport limit their deployment on a global scale, especially in areas with limited resources. The in vivo transfer of nucleic acid-based technologies encoding optimized therapeutic antibodies transform the body into a bioreactor for rapid and sustained production of biologics and hold great promise for circumventing the obstacles faced by the traditional delivery of antibodies. In this review, we provide an overview of the different antibody delivery strategies that are currently being developed, with particular emphasis on in vivo transfection of naked plasmid DNA facilitated by electroporation.

c-Abl-deficient mice exhibit reduced numbers of peritoneal B-1 cells and defects in BCR-induced B cell activation.

A role for c-Abl in B cell development and signaling has been suggested by previous work showing that c-Abl-deficient mice have defects in bone marrow B cell development and that c-Abl-deficient B cells are hypoproliferative in response to antigen receptor stimulation. Here we show that in addition to defects in early B cell development, c-Abl-deficient mice have defects in peripheral B cell development, including reduced percentages of peritoneal B-1 cells as well as transitional and marginal zone B cells in the spleen. It has been shown that c-Abl kinase activity increases upon B cell receptor (BCR) stimulation and that one of the targets of tyrosine phosphorylation by c-Abl is CD19. However, the consequences of c-Abl activity on B cell activation and CD19 signaling remain unknown. Here, we show that c-Abl-deficient splenic B cells exhibit reduced calcium flux in response to CD19 cross-linking, consistent with a role for c-Abl in CD19-dependent signaling. Additionally, we show that c-Abl-deficient B cells are defective in their ability to be activated in response to antigen receptor engagement, suggesting a functional role for c-Abl in BCR-dependent activation signaling pathways.

In vivo expressed biologics for infectious disease prophylaxis: rapid delivery of DNA-based antiviral antibodies.

With increasing frequency, humans are facing outbreaks of emerging infectious diseases (EIDs) with the potential to cause significant morbidity and mortality. In the most extreme instances, such outbreaks can become pandemics, as we are now witnessing with COVID-19. According to the World Health Organization, this new disease, caused by the novel coronavirus SARS-CoV-2, has already infected more than 10 million people worldwide and led to 499,913 deaths as of 29 June, 2020. How high these numbers will eventually go depends on many factors, including policies on travel and movement, availability of medical support, and, because there is no vaccine or highly effective treatment, the pace of biomedical research. Other than an approved antiviral drug that can be repurposed, monoclonal antibodies (mAbs) hold the most promise for providing a stopgap measure to lessen the impact of an outbreak while vaccines are in development. Technical advances in mAb identification, combined with the flexibility and clinical experience of mAbs in general, make them ideal candidates for rapid deployment. Furthermore, the development of mAb cocktails can provide a faster route to developing a robust medical intervention than searching for a single, outstanding mAb. In addition, mAbs are well-suited for integration into platform technologies for delivery, in which minimal components need to be changed in order to be redirected against a novel pathogen. In particular, utilizing the manufacturing and logistical benefits of DNA-based platform technologies in order to deliver one or more antiviral mAbs has the potential to revolutionize EID responses.

VSV-Displayed HIV-1 Envelope Identifies Broadly Neutralizing Antibodies Class-Switched to IgG and IgA.

The HIV-1 envelope (Env) undergoes conformational changes during infection. Broadly neutralizing antibodies (bNAbs) are typically isolated by using soluble Env trimers, which do not capture all Env states. To address these limitations, we devised a vesicular stomatitis virus (VSV)-based probe to display membrane-embedded Env trimers and isolated five bNAbs from two chronically infected donors, M4008 and M1214. Donor B cell receptor (BCR) repertoires identified two bNAb lineages, M4008_N1 and M1214_N1, that class-switched to immunoglobulin G (IgG) and IgA. Variants of these bNAbs reconstituted as IgA demonstrated broadly neutralizing activity, and the IgA fraction of M1214 plasma conferred neutralization. M4008_N1 epitope mapping revealed a glycan-independent V3 epitope conferring tier 2 virus neutralization. A 4.86-Å-resolution cryogenic electron microscopy (cryo-EM) structure of M1214_N1 complexed with CH505 SOSIP revealed another elongated epitope, the V2V5 corridor, extending from V2 to V5. Overall, the VSVENV probe identified bNAb lineages with neutralizing IgG and IgA members targeting distinct sites of HIV-1 Env vulnerability.

Rhabdo-immunodeficiency virus, a murine model of acute HIV-1 infection.

Numerous challenges have impeded HIV-1 vaccine development. Among these is the lack of a convenient small animal model in which to study antibody elicitation and efficacy. We describe a chimeric Rhabdo-Immunodeficiency virus (RhIV) murine model that recapitulates key features of HIV-1 entry, tropism and antibody sensitivity. RhIVs are based on vesicular stomatitis viruses (VSV), but viral entry is mediated by HIV-1 Env proteins from diverse HIV-1 strains. RhIV infection of transgenic mice expressing human CD4 and CCR5, exclusively on mouse CD4+ cells, at levels mimicking those on human CD4+ T-cells, resulted in acute, resolving viremia and CD4+ T-cell depletion. RhIV infection elicited protective immunity, and antibodies to HIV-1 Env that were primarily non-neutralizing and had modest protective efficacy following passive transfer. The RhIV model enables the convenient in vivo study of HIV-1 Env-receptor interactions, antiviral activity of antibodies and humoral responses against HIV-1 Env, in a genetically manipulatable host.