Vaccination induces broadly neutralizing antibody precursors to HIV gp41.

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

Biphasic and cardiomyocyte-specific IFIT activity protects cardiomyocytes from enteroviral infection.

Viral myocarditis is a serious disease, commonly caused by type B coxsackieviruses (CVB). Here we show that innate immune protection against CVB3 myocarditis requires the IFIT (IFN-induced with tetratricopeptide) locus, which acts in a biphasic manner. Using IFIT locus knockout (IFITKO) cardiomyocytes we show that, in the absence of the IFIT locus, viral replication is dramatically increased, indicating that constitutive IFIT expression suppresses CVB replication in this cell type. IFNß pre-treatment strongly suppresses CVB3 replication in wild type (wt) cardiomyocytes, but not in IFITKO cardiomyocytes, indicating that other interferon-stimulated genes (ISGs) cannot compensate for the loss of IFITs in this cell type. Thus, in isolated wt cardiomyocytes, the anti-CVB3 activity of IFITs is biphasic, being required for protection both before and after T1IFN signaling. These in vitro findings are replicated in vivo. Using novel IFITKO mice we demonstrate accelerated CVB3 replication in pancreas, liver and heart in the hours following infection. This early increase in virus load in IFITKO animals accelerates the induction of other ISGs in several tissues, enhancing virus clearance from some tissues, indicating that-in contrast to cardiomyocytes-other ISGs can offset the loss of IFITs from those cell types. In contrast, CVB3 persists in IFITKO hearts, and myocarditis occurs. Thus, cardiomyocytes have a specific, biphasic, and near-absolute requirement for IFITs to control CVB infection.

Type I IFN Signaling Is Dispensable during Secondary Viral Infection.

Innate immune responses in general, and type I interferons (T1IFNs) in particular, play an important and often essential role during primary viral infections, by directly combatting the virus and by maximizing the primary adaptive immune response. Several studies have suggested that T1IFNs also contribute very substantially to the secondary (recall) response; they are thought (i) to be required to drive the early attrition of memory T cells, (ii) to support the subsequent expansion of surviving virus-specific memory cells, and (iii) to assist in the suppression and clearance of the infectious agent. However, many of these observations were predicated upon models in which T1IFN signaling was interrupted prior to a primary immune response, raising the possibility that the resulting memory cells might be intrinsically abnormal. We have directly addressed this by using an inducible-Cre model system in which the host remains genetically-intact during the primary response to infection, and in which T1IFN signaling can be effectively ablated prior to secondary viral challenge. We report that, in stark contrast to primary infection, T1IFN signaling is not required during the recall response. IFNαßR-deficient memory CD8+ and CD4+ memory T cells undergo attrition and expansion with kinetics that are indistinguishable from those of receptor-sufficient cells. Moreover, even in the absence of functional T1IFN signaling, the host's immune capacity to rapidly suppress, and then to eradicate, a secondary infection remains intact. Thus, this study shows that T1IFN signaling is dispensable during the recall response to a virus infection. Moreover, two broader implications may be drawn. First, a T cell's requirement for a cytokine is highly dependent on the cell's maturation / differentiation status. Consequently, second, these data underscore the importance of evaluating a gene's impact by modulating its expression or function in a temporally-controllable manner.

Osteopontin Impacts West Nile virus Pathogenesis and Resistance by Regulating Inflammasome Components and Cell Death in the Central Nervous System at Early Time Points.

Osteopontin (OPN) is a molecule that is common in central nervous system (CNS) pathologies, which participates in the activation, migration, and survival of inflammatory cells. However, the mechanisms by which OPN modulates inflammatory pathways are not clear. To understand the role of OPN in CNS viral infections, we used a lethal mouse model of West Nile virus (WNV), characterized by the injection of high doses of the Eg101 strain of WNV, causing the increase of OPN levels in the brain since early time points. To measure the impact of OPN in neuropathogenesis and resistance, we compared C57BI/6 WT with mice lacking the OPN gene (OPN KO). OPN KO presented a significantly higher mortality compared to WT mice, detectable since day 5 pi. Our data suggests that OPN expression at early time points may provide protection against viral spread in the CNS by negatively controlling the type I IFN-sensitive, caspase 1-dependent inflammasome, while promoting an alternative caspase 8-associated pathway, to control the apoptosis of infected cells during WNV infection in the CNS. Overall, we conclude that the expression of OPN maintains a critical threshold in the innate immune response that controls apoptosis and lethal viral spread in early CNS infection.

Antigen-specific naive CD8+ T cells produce a single pulse of IFN-γ in vivo within hours of infection, but without antiviral effect.

In vitro studies have shown that naive CD8(+) T cells are unable to express most of their effector proteins until after at least one round of cell division has taken place. We have reassessed this issue in vivo and find that naive CD8(+) T cells mount Ag-specific responses within hours of infection, before proliferation has commenced. Newly activated naive Ag-specific CD8(+) T cells produce a rapid pulse of IFN-γ in vivo and begin to accumulate granzyme B and perforin. Later, in vivo cytolytic activity is detectable, coincident with the initiation of cell division. Despite the rapid development of these functional attributes, no antiviral effect was observed early during infection, even when the cells are present in numbers similar to those of virus-specific memory cells. The evolutionary reason for the pulse of IFN-γ synthesis by naive T cells is uncertain, but the lack of antiviral impact suggests that it may be regulatory.

CD8+ T cells maintain suppression of simian immunodeficiency virus in the central nervous system.

Human immunodeficiency virus (HIV) accesses the brain early in infection and can lead to neurocognitive disorders. The brain can also serve as a viral reservoir, but how virus is controlled in the brain is unknown. To examine this, CD8-depleting monoclonal antibody was injected into the cerebrospinal fluid of rhesus monkeys with chronic simian immunodeficiency virus (SIV) infection. This treatment led to the rapid increase of SIV in the brain. Virus in the brain is maintained by active suppression from the host immune system. This dynamic interaction can be manipulated in efforts to control and eradicate virus from the brain and other reservoirs.

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. Here, we investigated the role of T1IFNs in the heart using a mouse model in which the T1IFNR gene can be ablated in vivo, specifically in cardiomyocytes. We found that T1IFN signaling into cardiomyocytes contributed substantially to the suppression of viral replication and infectious virus yield in the heart; in the absence of such signaling, virus titers were markedly elevated by day 3 postinfection (p.i.) and remained high at day 12 p.i., a time point at which virus was absent from genetically intact littermates, suggesting that the T1IFN-unresponsive cardiomyocytes may act as a safe haven for the virus. Nevertheless, in these mice the myocardial infection remained highly focal, despite the cardiomyocytes' inability to respond to T1IFN, indicating that other factors, as yet unidentified, are sufficient to prevent the more widespread dissemination of the infection throughout the heart. The absence of T1IFN signaling into cardiomyocytes also was accompanied by a profound acceleration and exacerbation of myocarditis and by a significant increase in mortality. IMPORTANCE: Acute coxsackievirus B3 (CVB3) infection is one of the most common causes of acute myocarditis, a serious and sometimes fatal disease. To optimize treatment, it is vital that we identify the immune factors that limit virus spread in the heart and other organs. Type I interferons play a key role in controlling many virus infections, but it has been suggested that they may not directly impact CVB3 infection within the heart. Here, using a novel line of transgenic mice, we show that these cytokines signal directly into cardiomyocytes, limiting viral replication, myocarditis, and death.

CD8+ memory T cells appear exhausted within hours of acute virus infection.

CD8(+) memory T cells are abundant and are activated in a near-synchronous manner by infection, thereby providing a unique opportunity to evaluate the coordinate functional and phenotypic changes that occur in vivo within hours of viral challenge. Using two disparate virus challenges of mice, we show that splenic CD8(+) memory T cells rapidly produced IFN-γ in vivo; however, within 18-24 h, IFN-γ synthesis was terminated and remained undetectable for ≥ 48 h. A similar on/off response was observed in CD8(+) memory T cells in the peritoneal cavity. Cessation of IFN-γ production in vivo occurred despite the continued presence of immunostimulatory viral Ag, indicating that the initial IFN-γ response had been actively downregulated and that the cells had been rendered refractory to subsequent in vivo Ag contact. Downregulation of IFN-γ synthesis was accompanied by the upregulation of inhibitory receptor expression on the T cells, and ex vivo analyses using synthetic peptides revealed a concurrent hierarchical loss of cytokine responsiveness (IL-2, then TNF, then IFN-γ) taking place during the first 24 h following Ag contact. Thus, within hours of virus challenge, CD8(+) memory T cells display the standard hallmarks of T cell exhaustion, a phenotype that previously was associated only with chronic diseases and that is generally viewed as a gradually developing and pathological change in T cell function. Our data suggest that, instead, the "exhaustion" phenotype is a rapid and normal physiological T cell response.

Osteopontin expression in the brain triggers localized inflammation and cell death when immune cells are activated by pertussis toxin.

Upregulation of osteopontin (OPN) is a characteristic of central nervous system pathologies. However, the role of OPN in inflammation is still controversial, since it can both prevent cell death and induce the migration of potentially damaging inflammatory cells. To understand the role of OPN in inflammation and cell survival, we expressed OPN, utilizing an adenoviral vector, in the caudoputamen of mice deficient in OPN, using beta-galactosidase- (ß-gal-) expressing vector as control. The tissue pathology and the expression of proinflammatory genes were compared in both treatments. Interestingly, inflammatory infiltrate was only found when the OPN-vector was combined with a peripheral treatment with pertussis toxin (Ptx), which activated peripheral cells to express the OPN receptor CD44v6. Relative to ß-gal, OPN increased the levels of inflammatory markers, including IL13Rα1, CXCR3, and CD40L. In Ptx-treated OPN KOs, apoptotic TUNEL+ cells surrounding the OPN expression site increased, compared to ß-gal. Together, these results show that local OPN expression combined with a peripheral inflammatory stimulus, such as Ptx, may be implicated in the development of brain inflammation and induction of cell death, by driving a molecular pattern characteristic of cytotoxicity. These are characteristics of inflammatory pathologies of the CNS in which OPN upregulation is a hallmark.

Heterologous prime-boost vaccination drives early maturation of HIV broadly neutralizing antibody precursors in humanized mice.

A protective HIV vaccine will likely need to induce broadly neutralizing antibodies (bnAbs). Vaccination with the germline-targeting immunogen eOD-GT8 60mer adjuvanted with AS01B was found to induce VRC01-class bnAb precursors in 97% of vaccine recipients in the IAVI G001 phase 1 clinical trial; however, heterologous boost immunizations with antigens more similar to the native glycoprotein will be required to induce bnAbs. Therefore, we designed core-g28v2 60mer, a nanoparticle immunogen to be used as a first boost after eOD-GT8 60mer priming. We found, using a humanized mouse model approximating human conditions of VRC01-class precursor B cell diversity, affinity, and frequency, that both protein- and mRNA-based heterologous prime-boost regimens induced VRC01-class antibodies that gained key mutations and bound to near-native HIV envelope trimers lacking the N276 glycan. We further showed that VRC01-class antibodies induced by mRNA-based regimens could neutralize pseudoviruses lacking the N276 glycan. These results demonstrated that heterologous boosting can drive maturation toward VRC01-class bnAb development and supported the initiation of the IAVI G002 phase 1 trial testing mRNA-encoded nanoparticle prime-boost regimens.

Efficient isolation of rare B cells using next-generation antigen barcoding.

The ability to efficiently isolate antigen-specific B cells in high throughput will greatly accelerate the discovery of therapeutic monoclonal antibodies (mAbs) and catalyze rational vaccine development. Traditional mAb discovery is a costly and labor-intensive process, although recent advances in single-cell genomics using emulsion microfluidics allow simultaneous processing of thousands of individual cells. Here we present a streamlined method for isolation and analysis of large numbers of antigen-specific B cells, including next generation antigen barcoding and an integrated computational framework for B cell multi-omics. We demonstrate the power of this approach by recovering thousands of antigen-specific mAbs, including the efficient isolation of extremely rare precursors of VRC01-class and IOMA-class broadly neutralizing HIV mAbs.

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). We show that in pancreatic acinar cells, CVB3 induces the formation of abundant small autophagy-like vesicles and permits amphisome formation. However, the virus markedly, albeit incompletely, limits the fusion of autophagosomes (and/or amphisomes) with lysosomes, and, perhaps as a result, very large autophagy-related structures are formed within infected cells; we term these structures megaphagosomes. Ultrastructural analyses confirmed that double-membraned autophagy-like vesicles were present in infected pancreatic tissue and that the megaphagosomes were related to the autophagy pathway; they also revealed a highly organized lattice, the individual components of which are of a size consistent with CVB RNA polymerase; we suggest that this may represent a coxsackievirus replication complex. Thus, these in vivo studies demonstrate that CVB3 infection dramatically modifies autophagy in infected pancreatic acinar cells.

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. In contrast, the presentation of CVB3-encoded MHC class II epitopes is relatively unencumbered, and CVB3 induces in vivo CD4(+) T cell responses that are, by several criteria, phenotypically normal. The cells display an effector phenotype and mature into multi-functional CVB3-specific memory CD4(+) T cells that expand dramatically following challenge infection and rapidly differentiate into secondary effector cells capable of secreting multiple cytokines. Our findings have implications for the efficiency of antigen cross-presentation during coxsackievirus infection.

Methamphetamine increases brain viral load and activates natural killer cells in simian immunodeficiency virus-infected monkeys.

Methamphetamine (Meth) abuse increases risky behaviors that contribute to the spread of HIV infection. In addition, because HIV and Meth independently affect physiological systems including the central nervous system, HIV-induced disease may be more severe in drug users. We investigated changes in blood and brain viral load as well as differences in immune cells in chronically simian immunodeficiency virus-infected rhesus macaques that were either administered Meth or used as controls. Although Meth administration did not alter levels of virus in the plasma, viral load in the brain was significantly increased in Meth-treated animals compared with control animals. Meth treatment also resulted in an activation of natural killer cells. Given the prevalence of Meth use in HIV-infected and HIV at-risk populations, these findings reveal the likely untoward effects of Meth abuse in such individuals.

A food-responsive switch modulates TFEB and autophagy, and determines susceptibility to coxsackievirus infection and pancreatitis.

Almost a billion people worldwide are chronically undernourished. Herein, using a mouse model of coxsackievirus B3 (CVB3) infection, we report that a single day of food restriction (FR) markedly increases susceptibility to attenuated enterovirus infection, replication, and disease. These "pro-viral" effects, which are rapidly-reversed by the restoration of food, are mediated by several genes whose expression is altered by FR, and which support CVB3 replication. Central to this is TFEB, a protein whose expression and activation status are rapidly increased by FR. TFEB, which regulates the transcription of >100 genes involved in macroautophagy/autophagy and lysosomal biogenesis, responds similarly to both FR and CVB3 infection and plays a pivotal role in determining host susceptibility to CVB3. We propose that, by upregulating TFEB, FR generates an intracellular environment that is more hospitable to the incoming virus, facilitating its replication. This interplay between nutritional status and enterovirus replication has implications for human health and, perhaps, for the evolution of these viruses.Abbreviations: Atg/ATG: autophagy-related; CAR: Coxsackievirus and adenovirus receptor; Cas9: CRISPR associated protein 9; Cre: recombinase that causes recombination; CRISPR: clustered regularly interspaced short palindromic repeats; Ctsb/CTSB: cathepsin B; CVB3: coxsackievirus B3; DsRedCVB3: a recombinant CVB3 that encodes the Discosoma red fluorescent protein; EL: elastase; FR: food restriction; GFP: green fluorescent protein; gRNA: guide RNA; HBSS: Hanks Buffered Salt Solution; LYNUS: lysosomal nutrient sensing machinery; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFI: mean fluorescence intensity; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; Nluc: nanoluciferase; NlucCVB3: a recombinant CVB3 encoding nanoluciferase; pfu: plaque-forming unit(s); p.i.: post infection; rCVB: recombinant coxsackievirus B3; RPS6KB/p70S6K: ribosomal protein S6 kinase; RT: room temperature; siRNA: small interfering RNA; TFEB: transcription factor EB; tg: transgenic; TUBB: ß-tubulin; UNINF: uninfected; wrt: with respect to; WT: wild type.

An integrated systems analysis implicates EGR1 downregulation in simian immunodeficiency virus encephalitis-induced neural dysfunction.

Human immunodeficiency virus (HIV)-associated dementia (HAD) is a syndrome occurring in HIV-infected patients with advanced disease that likely develops as a result of macrophage and microglial activation as well as other immune events triggered by virus in the central nervous system. The most relevant experimental model of HAD, rhesus macaques exhibiting simian immunodeficiency virus (SIV) encephalitis (SIVE), closely reproduces the human disease and has been successfully used to advance our understanding of mechanisms underlying HAD. In this study we integrate gene expression data from uninfected and SIV-infected hippocampus with a human protein interaction network and discover modules of genes whose expression patterns distinguish these two states, to facilitate identification of neuronal genes that may contribute to SIVE/HIV cognitive deficits. Using this approach we identify several downregulated candidate genes and select one, EGR1, a key molecule in hippocampus-related learning and memory, for further study. We show that EGR1 is downregulated in SIV-infected hippocampus and that it can be downregulated in differentiated human neuroblastoma cells by treatment with CCL8, a product of activated microglia. Integration of expression data with protein interaction data to discover discriminatory modules of interacting proteins can be usefully used to prioritize differentially expressed genes for further study. Investigation of EGR1, selected in this manner, indicates that its downregulation in SIVE may occur as a consequence of the host response to infection, leading to deficits in cognition.

A technique for intracisternal collection and administration in a rhesus macaque.

Accessing the subarachnoid space via intrathecal needle placement can be done in nonhuman primates (NHPs) and other species to collect cerebral spinal fluid (CSF) or to deliver agents into the CSF. Researchers can use a variety of techniques for intrathecal collection and administration. Drawing on various published resources and previous experience with intracisternal CSF sampling, the authors sought to modify one collection technique to include CSF administration. Here, the authors describe how they collected CSF from the cisterna magna of rhesus macaques (Macaca mulatta) and administered substances through the cisterna magna into the CSF. They identify potential concerns that they had when developing the technique and describe how they alleviated those concerns. The authors found this technique, which relies heavily on teamwork, to be an effective method for intracisternal injection of an experimental compound.

Hepatocytes trap and silence coxsackieviruses, protecting against systemic disease in mice.

Previous research suggests that hepatocytes catabolize chemical toxins but do not remove microbial agents, which are filtered out by other liver cells (Kupffer cells and endothelial cells). Here we show that, contrary to current understanding, hepatocytes trap and rapidly silence type B coxsackieviruses (CVBs). In genetically wildtype mice, this activity causes hepatocyte damage, which is alleviated in mice carrying a hepatocyte-specific deletion of the coxsackievirus-adenovirus receptor. However, in these mutant mice, there is a dramatic early rise in blood-borne virus, followed by accelerated systemic disease and increased mortality. Thus, wild type hepatocytes act similarly to a sponge for CVBs, protecting against systemic illness at the expense of their own survival. We speculate that hepatocytes may play a similar role in other viral infections as well, thereby explaining why hepatocytes have evolved their remarkable regenerative capacity. Our data also suggest that, in addition to their many other functions, hepatocytes might be considered an integral part of the innate immune system.

Human immunodeficiency virus-1/surface glycoprotein 120 induces apoptosis through RNA-activated protein kinase signaling in neurons.

Previous work has demonstrated that the surface glycoprotein (gp120) of human immunodeficiency virus-1 (HIV-1) can induce damage and apoptosis of neurons both in vitro and in vivo. In this report, we provide evidence that double-stranded RNA-activated protein kinase (PKR), a stress kinase, is involved in HIV/gp120-associated neurodegeneration. In cultures of mixed cortical cells, HIV/gp120 increased the protein level of PKR. Additionally, PKR was phosphorylated in neurons but not glia after exposure to gp120. The use of two independent pharmacological inhibitors of PKR activity abrogated neuronal cell death induced by gp120. Cortical neurons from PKR knock-out mice were significantly protected from neurotoxicity induced by gp120, further validating the pivotal proapoptotic function of PKR. gp120-induced phosphorylated PKR localized prominently to neuronal nuclei; PKR inhibition or the NMDA receptor antagonist MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate] abrogated this effect. PKR inactivation also inhibited gp120-induced caspase-3 activation, consistent with its neuroprotective effect. Finally, brain tissue from individuals diagnosed with HIV-associated dementia (HAD), but not HIV infection alone, contained the activated form of PKR, which localized predominantly to neuronal nuclei. Together, these results identify PKR as a critical mediator of gp120 neurotoxicity, suggesting that activation of PKR contributes to the neuronal injury and cell death observed in HAD.