Physicochemical and structural insights into lyophilized mRNA-LNP from lyoprotectant and buffer screenings.

The surge in RNA therapeutics has revolutionized treatments for infectious diseases like COVID-19 and shows the potential to expand into other therapeutic areas. However, the typical requirement for ultra-cold storage of mRNA-LNP formulations poses significant logistical challenges for global distribution. Lyophilization serves as a potential strategy to extend mRNA-LNP stability while eliminating the need for ultra-cold supply chain logistics.

Nucleotide modifications enable rational design of TLR7-selective ligands by blocking RNase cleavage.

Toll-like receptors 7 (TLR7) and 8 (TLR8) each sense single-stranded RNA (ssRNA), but their activation results in different immune activation profiles. Attempts to selectively target either TLR7 or TLR8 have been hindered by their high degree of homology. However, recent studies revealed that TLR7 and TLR8 bind different ligands resulting from the processing of ssRNA by endolysosomal RNases.

In vivo ablation of type I interferon receptor from cardiomyocytes delays coxsackieviral clearance and accelerates myocardial disease.

Unlabelled: Acute coxsackievirus B3 (CVB3) infection is one of the most prevalent causes of acute myocarditis, a disease that frequently is identified only after the sudden death of apparently healthy individuals. CVB3 infects cardiomyocytes, but the infection is highly focal, even in the absence of a strong adaptive immune response, suggesting that virus spread within the heart may be tightly constrained by the innate immune system. Type I interferons (T1IFNs) are an obvious candidate, and T1IFN receptor (T1IFNR) knockout mice are highly susceptible to CVB3 infection, succumbing within a few days of challenge.

Wild-type coxsackievirus infection dramatically alters the abundance, heterogeneity, and immunostimulatory capacity of conventional dendritic cells in vivo.

In vitro studies have shown that enteroviruses employ strategies that may impair the ability of DCs to trigger T cell immunity, but it is unclear how these viruses affect DCs in vivo. Here, we evaluate the effects of wild-type (wt) coxsackievirus B3 on DCs in vitro and in a murine model in vivo. Although CVB3 does not productively infect the vast majority of DCs, virus infection profoundly reduces splenic conventional DC numbers and diminishes their capacity to prime naïve CD8(+) T cells in vitro.

Autophagy, inflammation and neurodegenerative disease.

Autophagy is emerging as a central regulator of cellular health and disease and, in the central nervous system (CNS), this homeostatic process appears to influence synaptic growth and plasticity. Herein, we review the evidence that dysregulation of autophagy may contribute to several neurodegenerative diseases of the CNS. Up-regulation of autophagy may prevent, delay or ameliorate at least some of these disorders, and - based on recent findings from our laboratory - we speculate that this goal may be achieved using a safe, simple and inexpensive approach.

Coxsackievirus infection induces autophagy-like vesicles and megaphagosomes in pancreatic acinar cells in vivo.

Autophagy can play an important part in protecting host cells during virus infection, and several viruses have developed strategies by which to evade or even exploit this homeostatic pathway. Tissue culture studies have shown that poliovirus, an enterovirus, modulates autophagy. Herein, we report on in vivo studies that evaluate the effects on autophagy of coxsackievirus B3 (CVB3).

Type B coxsackieviruses and their interactions with the innate and adaptive immune systems.

Coxsackieviruses are important human pathogens, and their interactions with the innate and adaptive immune systems are of particular interest. Many viruses evade some aspects of the innate response, but coxsackieviruses go a step further by actively inducing, and then exploiting, some features of the host cell response. Furthermore, while most viruses encode proteins that hinder the effector functions of adaptive immunity, coxsackieviruses and their cousins demonstrate a unique capacity to almost completely evade the attention of naive CD8(+) T cells.

Heterogeneity among viral antigen-specific CD4+ T cells and their de novo recruitment during persistent polyomavirus infection.

Virus-specific CD4(+) T cells optimize antiviral responses by providing help for antiviral humoral responses and CD8(+) T cell differentiation. Although CD4(+) T cell responses to viral infections that undergo complete clearance have been studied extensively, less is known about virus-specific CD4(+) T cell responses to viruses that persistently infect their hosts. Using a mouse polyomavirus (MPyV) infection model, we previously demonstrated that CD4(+) T cells are essential for recruiting naive MPyV-specific CD8(+) T cells in persistently infected mice.

Short-term fasting induces profound neuronal autophagy.

Disruption of autophagy--a key homeostatic process in which cytosolic components are degraded and recycled through lysosomes--can cause neurodegeneration in tissue culture and in vivo. Upregulation of this pathway may be neuroprotective, and much effort is being invested in developing drugs that cross the blood brain barrier and increase neuronal autophagy. One well-recognized way of inducing autophagy is by food restriction, which upregulates autophagy in many organs including the liver; but current dogma holds that the brain escapes this effect, perhaps because it is a metabolically privileged site.

Coxsackievirus B3 inhibits antigen presentation in vivo, exerting a profound and selective effect on the MHC class I pathway.

Many viruses encode proteins whose major function is to evade or disable the host T cell response. Nevertheless, most viruses are readily detected by host T cells, and induce relatively strong T cell responses. Herein, we employ transgenic CD4(+) and CD8(+) T cells as sensors to evaluate in vitro and in vivo antigen presentation by coxsackievirus B3 (CVB3), and we show that this virus almost completely inhibits antigen presentation via the MHC class I pathway, thereby evading CD8(+) T cell immunity.

Enumeration and functional evaluation of virus-specific CD4+ and CD8+ T cells in lymphoid and peripheral sites of coxsackievirus B3 infection.

Previous studies have suggested that coxsackievirus B (CVB) activates CD8(+) T cells in vivo, but the extent of this activation and the antigen specificity of the CD8(+) T cells remain uncertain. Furthermore, CVB-induced CD4(+) T-cell responses have not been carefully investigated. Herein, we evaluate CD8(+) and CD4(+) T-cell responses both in a secondary lymphoid organ (spleen) and in peripheral tissues (heart and pancreas), using a recombinant CVB3 (rCVB3.

Allogeneic differences in the dependence on CD4+ T-cell help for virus-specific CD8+ T-cell differentiation.

CD4(+) T-cell help enables antiviral CD8(+) T cells to differentiate into fully competent memory cells and sustains CD8(+) T-cell-mediated immunity during persistent virus infection. We recently reported that mice of C57BL/6 and C3H strains differ in their dependence on CD28 and CD40L costimulation for long-term control of infection by polyoma virus, a persistent mouse pathogen. In this study, we asked whether mice of these inbred strains also vary in their requirement for CD4(+) T-cell help for generating and maintaining polyoma virus-specific CD8(+) T cells.

The antiviral CD8+ T cell response is differentially dependent on CD4+ T cell help over the course of persistent infection.

Although many studies have investigated the requirement for CD4(+) T cell help for CD8(+) T cell responses to acute viral infections that are fully resolved, less is known about the role of CD4(+) T cells in maintaining ongoing CD8(+) T cell responses to persistently infecting viruses. Using mouse polyoma virus (PyV), we asked whether CD4(+) T cell help is required to maintain antiviral CD8(+) T cell and humoral responses during acute and persistent phases of infection. Though fully intact during acute infection, the PyV-specific CD8(+) T cell response declined numerically during persistent infection in MHC class II-deficient mice, leaving a small antiviral CD8(+) T cell population that was maintained long term.

Continuous recruitment of naive T cells contributes to heterogeneity of antiviral CD8 T cells during persistent infection.

Numerous microbes establish persistent infections, accompanied by antigen-specific CD8 T cell activation. Pathogen-specific T cells in chronically infected hosts are often phenotypically and functionally variable, as well as distinct from T cells responding to nonpersistent infections; this phenotypic heterogeneity has been attributed to an ongoing reencounter with antigen. Paradoxically, maintenance of memory CD8 T cells to acutely resolved infections is antigen independent, whereas there is a dependence on antigen for T cell survival in chronically infected hosts.

Costimulation requirements for antiviral CD8+ T cells differ for acute and persistent phases of polyoma virus infection.

The requirement for costimulation in antiviral CD8+ T cell responses has been actively investigated for acutely resolved viral infections, but it is less defined for CD8+ T cell responses to persistent virus infection. Using mouse polyoma virus (PyV) as a model of low-level persistent virus infection, we asked whether blockade of the CD40 ligand (CD40L) and CD28 costimulatory pathways impacts the magnitude and function of the PyV-specific CD8+ T response, as well as the humoral response and viral control during acute and persistent phases of infection. Costimulation blockade or gene knockout of either CD28 or CD40L substantially dampened the magnitude of the acute CD8+ T cell response; simultaneous CD28 and CD40L blockade severely depressed the acute T cell response, altered the cell surface phenotype of PyV-specific CD8+ T cells, decreased PyV VP1-specific serum IgG titers, and resulted in an increase in viral DNA levels in multiple organs.

Late priming and variability of epitope-specific CD8+ T cell responses during a persistent virus infection.

Control of persistently infecting viruses requires that antiviral CD8(+) T cells sustain their numbers and effector function. In this study, we monitored epitope-specific CD8(+) T cells during acute and persistent phases of infection by polyoma virus, a mouse pathogen that is capable of potent oncogenicity. We identified several novel polyoma-specific CD8(+) T cell epitopes in C57BL/6 mice, a mouse strain highly resistant to polyoma virus-induced tumors.

Cutting edge: rapid in vivo CTL activity by polyoma virus-specific effector and memory CD8+ T cells.

For viruses that establish persistent infection, continuous immunosurveillance by effector-competent antiviral CD8(+) T cells is likely essential for limiting viral replication. Although it is well documented that virus-specific memory CD8(+) T cells synthesize cytokines after short term in vitro stimulation, there is limited evidence that these T cells exhibit cytotoxicity, the dominant antiviral effector function. Here, we show that antiviral CD8(+) T cells in mice acutely infected by polyoma virus, a persistent mouse pathogen, specifically eliminate viral peptide-pulsed donor spleen cells within minutes after adoptive transfer and do so via a perforin-dependent mechanism.

Regulation of antiviral CD8+ T cells by inhibitory natural killer cell receptors.

Recent evidence indicates that CD8(+) T cells express natural killer cell receptors that constrain the range and magnitude of their activities. For virus-specific CD8(+) T cells, upregulation of these receptors serves to control infection, while concurrently minimizing bystander pathology. Dysregulated expression of these receptors, however, may foster the establishment of persistent virus infection.

Peptide-independent folding and CD8 alpha alpha binding by the nonclassical class I molecule, thymic leukemia antigen.

The nonclassical class I molecule, thymic leukemia (TL), has been shown to be expressed on intestinal epithelial cells and to interact with CD8(+) intraepithelial T lymphocytes. We generated recombinant soluble TL (T18(d)) H chains in bacteria as inclusion bodies and refolded them with beta(2)-microglobulin in the presence or absence of a random peptide library. Using a mAb, HD168, that recognizes a conformational epitope on native TL molecules, we observed that protein folds efficiently in the absence of peptide.