An AGS-associated mutation in ADAR1 catalytic domain results in early-onset and MDA5-dependent encephalopathy with IFN pathway activation in the brain.

BACKGROUND: Aicardi-Goutières syndrome (AGS) is a severe neurodegenerative disease with clinical features of early-onset encephalopathy and progressive loss of intellectual abilities and motor control. Gene mutations in seven protein-coding genes have been found to be associated with AGS. However, the causative role of these mutations in the early-onset neuropathogenesis has not been demonstrated in animal models, and the mechanism of neurodegeneration of AGS remains ambiguous. METHODS: Via CRISPR/Cas-9 technology, we established a mutant mouse model in which a genetic mutation found in AGS patients at the ADAR1 coding gene (Adar) loci was introduced into the mouse genome. A mouse model carrying double gene mutations encoding ADAR1 and MDA-5 was prepared using a breeding strategy. Phenotype, gene expression, RNA sequencing, innate immune pathway activation, and pathologic studies including RNA in situ hybridization (ISH) and immunohistochemistry were used for characterization of the mouse models to determine potential disease mechanisms. RESULTS: We established a mouse model bearing a mutation in the catalytic domain of ADAR1, the D1113H mutation found in AGS patients. With this mouse model, we demonstrated a causative role of this mutation for the early-onset brain injuries in AGS and determined the signaling pathway underlying the neuropathogenesis. First, this mutation altered the RNA editing profile in neural transcripts and led to robust IFN-stimulated gene (ISG) expression in the brain. By ISH, the brains of mutant mice showed an unusual, multifocal increased expression of ISGs that was cell-type dependent. Early-onset astrocytosis and microgliosis and later stage calcification in the deep white matter areas were observed in the mutant mice. Brain ISG activation and neuroglial reaction were completely prevented in the Adar D1113H mutant mice by blocking RNA sensing through deletion of the cytosolic RNA receptor MDA-5. CONCLUSIONS: The Adar D1113H mutation in the ADAR1 catalytic domain results in early-onset and MDA5-dependent encephalopathy with IFN pathway activation in the mouse brain.

Gamma Knife Radiosurgery in the Management of Hypothalamic Glioma: A Case Report with Long-Term Follow-Up.

BACKGROUND: Optic pathway/hypothalamic gliomas are rare pediatric brain tumors. The management paradigm for these challenging tumors includes chemotherapy, radiotherapy, or surgical resection, but the optimal management strategy remains elusive. Gamma knife radiosurgery (GKRS) has emerged as a promising treatment for such lesions as documented by a small number of cases in the literature. CASE PRESENTATION: We present a rare case of hypothalamic glioma in a 13-year-old girl who was referred to our service due to growth of an incidentally diagnosed hypothalamic lesion following head injury at the age of 8 years. The lesion demonstrated hypointensity on T1- and hyperintensity on T2-weighted imaging without contrast enhancement. Given the growth of the lesion on serial imaging, a stereotactic biopsy was performed demonstrating low-grade glioma. The patient underwent GKRS treatment with a marginal dose of 15 Gy at 50% isodose line for a tumor volume of 2.2 mL. Annual radiological surveillance over the next 17 years demonstrated a gradual shrinkage of the lesion until it completely disappeared. The patient is currently a healthy 31-year-old female without any visual, endocrine, or neurocognitive deficits. CONCLUSION: The outcome obtained after extended follow-up in our patient highlights the safety and efficacy of GKRS in the management of hypothalamic gliomas in pediatrics, which in turn can avoid potentially serious complications of surgery in this vulnerable patient population, especially in this sensitive location.

Aicardi-Goutières syndrome-associated mutation at ADAR1 gene locus activates innate immune response in mouse brain.

BACKGROUND: Aicardi-Goutières syndrome (AGS) is a severe infant or juvenile-onset autoimmune disease characterized by inflammatory encephalopathy with an elevated type 1 interferon-stimulated gene (ISG) expression signature in the brain. Mutations in seven different protein-coding genes, all linked to DNA/RNA metabolism or sensing, have been identified in AGS patients, but none of them has been demonstrated to activate the IFN pathway in the brain of an animal. The molecular mechanism of inflammatory encephalopathy in AGS has not been well defined. Adenosine Deaminase Acting on RNA 1 (ADAR1) is one of the AGS-associated genes. It carries out A-to-I RNA editing that converts adenosine to inosine at double-stranded RNA regions. Whether an AGS-associated mutation in ADAR1 activates the IFN pathway and causes autoimmune pathogenesis in the brain is yet to be determined. METHODS: Mutations in the ADAR1 gene found in AGS patients were introduced into the mouse genome via CRISPR/Cas9 technology. Molecular activities of the specific p.K999N mutation were investigated by measuring the RNA editing levels in brain mRNA substrates of ADAR1 through RNA sequencing analysis. IFN pathway activation in the brain was assessed by measuring ISG expression at the mRNA and protein level through real-time RT-PCR and Luminex assays, respectively. The locations in the brain and neural cell types that express ISGs were determined by RNA in situ hybridization (ISH). Potential AGS-related brain morphologic changes were assessed with immunohistological analysis. Von Kossa and Luxol Fast Blue staining was performed on brain tissue to assess calcification and myelin, respectively. RESULTS: Mice bearing the ADAR1 p.K999N were viable though smaller than wild type sibs. RNA sequencing analysis of neuron-specific RNA substrates revealed altered RNA editing activities of the mutant ADAR1 protein. Mutant mice exhibited dramatically elevated levels of multiple ISGs within the brain. RNA ISH of brain sections showed selective activation of ISG expression in neurons and microglia in a patchy pattern. ISG-15 mRNA was upregulated in ADAR1 mutant brain neurons whereas CXCL10 mRNA was elevated in adjacent astroglia. No calcification or gliosis was detected in the mutant brain. CONCLUSIONS: We demonstrated that an AGS-associated mutation in ADAR1, specifically the p.K999N mutation, activates the IFN pathway in the mouse brain. The ADAR1 p.K999N mutant mouse replicates aspects of the brain interferonopathy of AGS. Neurons and microglia express different ISGs. Basal ganglia calcification and leukodystrophy seen in AGS patients were not observed in K999N mutant mice, indicating that development of the full clinical phenotype may need an additional stimulus besides AGS mutations. This mutant mouse presents a robust tool for the investigation of AGS and neuroinflammatory diseases including the modeling of potential "second hits" that enable severe phenotypes of clinically variable diseases.

Age-Related Pathology Associated with H1N1 A/California/07/2009 Influenza Virus Infection.

Influenza virus infection causes a spectrum of diseases, ranging from mild upper respiratory tract infection to severe lower respiratory tract infection, that can lead to diffuse alveolar damage, interstitial and airspace inflammation, or acute respiratory failure. Mechanisms instructing disease severity are not completely understood, but host, viral, and bacterial factors influence disease outcome. With age being one host factor associated with a higher risk of severe influenza, we investigated regional pulmonary distribution and severity of pneumonia after 2009 H1N1 influenza virus infection in newly weaned, adult, and aged ferrets to better understand age-dependent susceptibility and pathology. Aged ferrets exhibited greater weight loss and higher rates of mortality than adult ferrets, whereas most newly weaned ferrets did not lose weight but had a lack of weight gain. Newly weaned ferrets exhibited minimal pneumonia, whereas adult and aged ferrets had a spectrum of pneumonia severity. Influenza virus-induced pneumonia peaked earliest in adult ferrets, whereas aged ferrets had delayed presentation. Bronchial severity differed among groups, but bronchial pathology was comparable among all cohorts. Alveolar infection was strikingly different among groups. Newly weaned ferrets had little alveolar cell infection. Adult and aged ferrets had alveolar infection, but aged ferrets were unable to clear infection. These different age-related pneumonia and infection patterns suggest therapeutic strategies to treat influenza should be tailored contingent on age.

Histologic Appearance of Iatrogenic Obstructive Hydrocephalus in the Fetal Lamb Model.

INTRODUCTION: Documentation of histologic findings associated with congenital hydrocephalus in the fetal lamb model is a critical step in evaluating the efficacy of ventriculoamniotic shunting in the human fetus. METHODS: Four fetal sheep had hydrocephalus induced at approximately 95 days' gestation. Two co-twins remained as controls. The ewes were euthanized at term. The lamb brains were fixed in formalin, paraffin-embedded, stained, and analyzed for markers of neuropathology. Astrocytosis, microgliosis, and axonal loss were assessed with immunocytochemistry for glial fibrillary acidic protein, ionized calcium-binding adapter, and neurofilament/amyloid precursor protein, respectively. Cortical gray matter extracellular matrix was assessed with staining for the lectin Wisteria Floribunda agglutinin. RESULTS: Hydrocephalic lamb brains demonstrated deep white matter damage with loss of projecting axonal tracts in regions physically distorted by hydrocephalus, similar to that seen in hydrocephalic humans. There was no evidence of abnormal neocortical neuronal migration; however, there was evidence for delayed maturation of the neocortical gray matter, possibly from increased intracerebral pressure and subsequent ischemia. Control lamb brains demonstrated none of the above findings. CONCLUSION: This histological approach can be used to further define the mechanism of brain damage associated with hydrocephalus and interpret the efficacy of ventriculoamniotic shunting on fetal lamb brain neuroanatomy.

Zika Virus infection of rhesus macaques leads to viral persistence in multiple tissues.

Zika virus (ZIKV), an emerging flavivirus, has recently spread explosively through the Western hemisphere. In addition to symptoms including fever, rash, arthralgia, and conjunctivitis, ZIKV infection of pregnant women can cause microcephaly and other developmental abnormalities in the fetus. We report herein the results of ZIKV infection of adult rhesus macaques. Following subcutaneous infection, animals developed transient plasma viremia and viruria from 1-7 days post infection (dpi) that was accompanied by the development of a rash, fever and conjunctivitis. Animals produced a robust adaptive immune response to ZIKV, although systemic cytokine response was minimal. At 7 dpi, virus was detected in peripheral nervous tissue, multiple lymphoid tissues, joints, and the uterus of the necropsied animals. Notably, viral RNA persisted in neuronal, lymphoid and joint/muscle tissues and the male and female reproductive tissues through 28 to 35 dpi. The tropism and persistence of ZIKV in the peripheral nerves and reproductive tract may provide a mechanism of subsequent neuropathogenesis and sexual transmission.

Correction: Zika Virus infection of rhesus macaques leads to viral persistence in multiple tissues.

[This corrects the article DOI: 10.1371/journal.ppat.1006219.].

Widespread Virus Replication in Alveoli Drives Acute Respiratory Distress Syndrome in Aerosolized H5N1 Influenza Infection of Macaques.

Human infections with highly pathogenic avian influenza A (H5N1) virus are frequently fatal but the mechanisms of disease remain ill-defined. H5N1 infection is associated with intense production of proinflammatory cytokines, but whether this cytokine storm is the main cause of fatality or is a consequence of extensive virus replication that itself drives disease remains controversial. Conventional intratracheal inoculation of a liquid suspension of H5N1 influenza virus in nonhuman primates likely results in efficient clearance of virus within the upper respiratory tract and rarely produces severe disease. We reasoned that small particle aerosols of virus would penetrate the lower respiratory tract and blanket alveoli where target cells reside. We show that inhalation of aerosolized H5N1 influenza virus in cynomolgus macaques results in fulminant pneumonia that rapidly progresses to acute respiratory distress syndrome with a fatal outcome reminiscent of human disease. Molecular imaging revealed intense lung inflammation coincident with massive increases in proinflammatory proteins and IFN-α in distal airways. Aerosolized H5N1 exposure decimated alveolar macrophages, which were widely infected and caused marked influx of interstitial macrophages and neutrophils. Extensive infection of alveolar epithelial cells caused apoptosis and leakage of albumin into airways, reflecting loss of epithelial barrier function. These data establish inhalation of aerosolized virus as a critical source of exposure for fatal human infection and reveal that direct viral effects in alveoli mediate H5N1 disease. This new nonhuman primate model will advance vaccine and therapeutic approaches to prevent and treat human disease caused by highly pathogenic avian influenza viruses.

Aged Chinese-origin rhesus macaques infected with SIV develop marked viremia in absence of clinical disease, inflammation or cognitive impairment.

BACKGROUND: Damage to the central nervous system during HIV infection can lead to variable neurobehavioral dysfunction termed HIV-associated neurocognitive disorders (HAND). There is no clear consensus regarding the neuropathological or cellular basis of HAND. We sought to study the potential contribution of aging to the pathogenesis of HAND. Aged (range = 14.7-24.8 year) rhesus macaques of Chinese origin (RM-Ch) (n = 23) were trained to perform cognitive tasks. Macaques were then divided into four groups to assess the impact of SIVmac251 infection (n = 12) and combined antiretroviral therapy (CART) (5 infected; 5 mock-infected) on the execution of these tasks. RESULTS: Aged SIV-infected RM-Ch demonstrated significant plasma viremia and modest CSF viral loads but showed few clinical signs, no elevations of systemic temperature, and no changes in activity levels, platelet counts or weight. Concentrations of biomarkers of acute and chronic inflammation such as soluble CD14, CXCL10, IL-6 and TNF-α are known to be elevated following SIV infection of young adult macaques of several species, but concentrations of these biomarkers did not shift after SIV infection in aged RM-Ch and remained similar to mock-infected macaques. Neither acute nor chronic SIV infection or CART had a significant impact on accuracy, speed or percent completion in a sensorimotor test. CONCLUSIONS: Viremia in the absence of a chronic elevated inflammatory response seen in some aged RM-Ch is reminiscent of SIV infection in natural disease resistant hosts. The absence of cognitive impairment during SIV infection in aged RM-Ch might be in part attributed to diminishment of some facets of the immunological response. Additional study encompassing species and age differences is necessary to substantiate this hypothesis.

A population neuroscience approach to the study of cerebral small vessel disease in midlife and late life: an invited review.

Aging in later life engenders numerous changes to the cerebral microvasculature. Such changes can remain clinically silent but are associated with greater risk for negative health outcomes over time. Knowledge is limited about the pathogenesis, prevention, and treatment of potentially detrimental changes in the cerebral microvasculature that occur with advancing age. In this review, we summarize literature on aging of the cerebral microvasculature, and we propose a conceptual framework to fill existing research gaps and advance future work on this heterogeneous phenomenon. We propose that the major gaps in this area are attributable to an incomplete characterization of cerebrovascular pathology, the populations being studied, and the temporality of exposure to risk factors. Specifically, currently available measures of age-related cerebral microvasculature changes are indirect, primarily related to parenchymal damage rather than direct quantification of small vessel damage, limiting the understanding of cerebral small vessel disease (cSVD) itself. Moreover, studies seldom account for variability in the health-related conditions or interactions with risk factors, which are likely determinants of cSVD pathogenesis. Finally, study designs are predominantly cross-sectional and/or have relied on single time point measures, leaving no clear evidence of time trajectories of risk factors or of change in cerebral microvasculature. We argue that more resources should be invested in 1) developing methodological approaches and basic science models to better understand the pathogenic and etiological nature of age-related brain microvascular diseases and 2) implementing state-of-the-science population study designs that account for the temporal evolution of cerebral microvascular changes in diverse populations across the lifespan.

The spectrum of neuropathological changes associated with congenital Zika virus infection.

A major concern associated with ZIKV infection is the increased incidence of microcephaly with frequent calcifications in infants born from infected mothers. To date, postmortem analysis of the central nervous system (CNS) in congenital infection is limited to individual reports or small series. We report a comprehensive neuropathological study in ten newborn babies infected with ZIKV during pregnancy, including the spinal cords and dorsal root ganglia (DRG), and also muscle, pituitaries, eye, systemic organs, and placentas. Using in situ hybridization (ISH) and electron microscopy, we investigated the role of direct viral infection in the pathogenesis of the lesions. Nine women had Zika symptoms between the 4th and 18th and one in the 28th gestational week. Two babies were born at 32, one at 34 and 36 weeks each and six at term. The cephalic perimeter was reduced in four, and normal or enlarged in six patients, although the brain weights were lower than expected. All had arthrogryposis, except the patient infected at 28 weeks gestation. We defined three patterns of CNS lesions, with different patterns of destructive, calcification, hypoplasia, and migration disturbances. Ventriculomegaly was severe in the first pattern due to midbrain damage with aqueduct stenosis/distortion. The second pattern had small brains and mild/moderate (ex-vacuo) ventriculomegaly. The third pattern, a well-formed brain with mild calcification, coincided with late infection. The absence of descending fibres resulted in hypoplastic basis pontis, pyramids, and cortico-spinal tracts. Spinal motor cell loss explained the intrauterine akinesia, arthrogryposis, and neurogenic muscle atrophy. DRG, dorsal nerve roots, and columns were normal. Lympho-histiocytic inflammation was mild. ISH showed meningeal, germinal matrix, and neocortical infection, consistent with neural progenitors death leading to proliferation and migration disorders. A secondary ischemic process may explain the destructive lesions. In conclusion, we characterized the destructive and malformative consequences of ZIKV in the nervous system, as reflected in the topography and severity of lesions, anatomic localization of the virus, and timing of infection during gestation. Our findings indicate a developmental vulnerability of the immature CNS, and shed light on possible mechanisms of brain injury of this newly recognized public health threat.

Human Zika and West Nile virus neurological infections: What is the difference?

The recent epidemic of West Nile Virus (WNV) infection in the United States was associated with severe neurological disease in immunocompromised hosts, while the emergence of Zika virus infection in the Americas has been notable for an association with increased microcephaly in the fetuses of infected mothers. Rare autopsies of WNV infected humans have shown multiple organ involvement with a clear neurotropism. We have recently had the opportunity to examine the distribution of Zika virus in autopsies of newborns from infected pregnancies. While both viruses infect multiple organs, Zika appears to cause neurological disease in the fetus through two different mechanisms. Infection during the first trimester showed the potential to infect neural progenitor cells causing severe developmental abnormalities, while infection later in gestation was associated with meningeal infection and destructive ischemic lesions of the brain. Both viruses infect kidney tubules but Zika shares a prominent hepatotropism characteristic of other flaviviruses (e.g., Dengue). Limited transplacental Zika infection would be consistent with restriction to primary maternal infections with high viremia. In the absence of a vaccine, restriction of travel by immunosuppressed and pregnant non-immune individuals to endemic regions seems prudent.

H1N1, but not H3N2, influenza A virus infection protects ferrets from H5N1 encephalitis.

UNLABELLED: Seasonal influenza causes substantial morbidity and mortality because of efficient human-to-human spread. Rarely, zoonotic strains of influenza virus spread to humans, where they have the potential to mediate new pandemics with high mortality. We studied systemic viral spread after intranasal infection with highly pathogenic avian influenza virus (H5N1 [A/Viet Nam/1203/2004]) in ferrets with or without prior pandemic H1N1pdm09 (A/Mexico/4108/2009) or H3N2 (A/Victoria/361/2011) infection. After intranasal challenge with H5N1 influenza virus, naive ferrets rapidly succumbed to systemic infection. Animals challenged with H5N1 influenza virus greater than 3 months after recovering from an initial H1N1pdm09 infection survived H5N1 virus challenge and cleared virus from the respiratory tract 4 days after infection. However, a prolonged low-level infection of hematopoietic elements in the small bowel lamina propria, liver, and spleen was present for greater than 2 weeks postinfection, raising the potential for reassortment of influenza genes in a host infected with multiple strains of influenza. Animals previously infected with an H3N2 influenza virus succumbed to systemic disease and encephalitis after H5N1 virus challenge. These results indicate prior infection with different seasonal influenza strains leads to radically different protection from H5N1 challenge and fatal encephalitis. IMPORTANCE: Seasonal influenza is efficiently transmitted from human to human, causing substantial morbidity and mortality. Rarely, zoonotic strains of influenza virus spread to humans, where they have the potential to mediate new pandemics with high mortality. Infection of naive ferrets with H5N1 avian influenza virus causes a rapid and lethal systemic disease. We studied systemic H5N1 viral spread after infection of ferrets with or without prior exposure to either of two seasonal influenza virus strains, H1N1 and H3N2. Ferrets previously infected with H1N1 survive H5N1 challenge while those previously infected with H3N2 die of encephalitis. However ferrets protected from lethal H5N1 infection develop persistent low-level infection of the small intestine, liver, or spleen, providing a nidus for future viral strain recombination. The mechanism by which prior infection with specific strains of seasonal influenza virus protect from lethal H5N1 challenge needs to be elucidated in order to design effective immunization and treatments.

Role for mammalian chitinase 3-like protein 1 in traumatic brain injury.

Traumatic brain injury (TBI) is accompanied by inflammatory infiltrates and CNS tissue response. The astrocytosis associated with TBI has been proposed to have both beneficial and detrimental effects on surviving neural tissue. We recently observed prominent astrocytic expression of YKL-40/chitinase 3-like protein 1 (CHI3L1) associated with severity of brain injury. The physiological role of CHI3L1 in the CNS is unknown; however, its distribution at the perimeter of contusions and temporal course of expression suggested that in TBI it might be an important component of the astrocytic response to modulate CNS inflammation. To address this hypothesis, we used serially sectioned brains to quantitatively compare the neuropathological outcomes of TBI produced by controlled cortical impact in wild type (WT) and chi3l1 knockout (KO) mice where the murine YKL-40 homologue, breast regression protein 39 (BRP-39/CHI3l1), had been homozygously disrupted. At 21 days post-injury, chi3l1 KO mice displayed greater astrocytosis (increased GFAP staining) in the hemispheres ipsilateral and contralateral to impact compared with WT mice. Similarly, Iba1 expression as a measure of microglial/macrophage response was significantly increased in chi3l1 KO compared with WT in the hemisphere contralateral to impact. We conclude that astrocytic expression of CHI3L1 limits the extent of both astrocytic and microglial/macrophage facets of neuroinflammation and suggests a novel potential therapeutic target for modulating neuroinflammation.

Coxsackievirus B4 myocarditis and meningoencephalitis in newborn twins.

Coxsackievirus B4 (CB4) is a picornavirus associated with a variety of human diseases, including neonatal meningoencephalitis, myocarditis and type 1 diabetes. We report the pathological findings in twin newborns who died during an acute infection. The twins were born 1 month premature but were well and neurologically intact at birth. After a week they developed acute lethal neonatal sepsis and seizures. Histopathology demonstrated meningoencephalitis and severe myocarditis, as well as pancreatitis, adrenal medullitis and nephritis. Abundant CB4 sequences were identified in nucleic acid extracted from the brain and heart. In situ hybridization with probes to CB4 demonstrated infection of neurons, myocardiocytes, endocrine pancreas and adrenal medulla. The distribution of infected cells and immune response is consistent with reported clinical symptomatology where systemic and neurological diseases are the result of CB4 infection of select target cells.

Molecular imaging of microglia/macrophages in the brain.

Neuroinflammation perpetuates neuronal damage in many neurological disorders. Activation of resident microglia and infiltration of monocytes/macrophages contributes to neuronal injury and synaptic damage. Noninvasive imaging of these cells in vivo provides a means to monitor progression of disease as well as assess efficacies of potential therapeutics. This review provides an overview of positron emission tomography (PET) and magnetic resonance (MR) imaging of microglia/macrophages in the brain. We describe the rationale behind PET imaging of microglia/macrophages with ligands that bind to translocator protein-18 kDa (TSPO). We discuss the prototype TSPO radioligand [(11)C]PK11195, its limitations, and the development of newer TSPO ligands as PET imaging agents. PET imaging agents for targets other than TSPO are emerging, and we outline the potential of these agents for imaging brain microglia/macrophage activity in vivo. Finally, we briefly summarize advances in MR imaging of microglia/macrophages using iron oxide nanoparticles and ultra-small super paramagnetic particles that are phagocytosed. Despite many technical advances, more sensitive agents are required to be useful indicators of neuroinflammation in brain.

Elicitation of anti-1918 influenza virus immunity early in life prevents morbidity and lower levels of lung infection by 2009 pandemic H1N1 influenza virus in aged mice.

The Spanish influenza virus pandemic of 1918 was responsible for 40 million to 50 million deaths and is antigenically similar to the swine lineage 2009 pandemic influenza virus. Emergence of the 2009 pandemic from swine into humans has raised the possibility that low levels of cross-protective immunity to past shared epitopes could confer protection. In this study, influenza viruslike particles (VLPs) were engineered to express the hemagglutinin (HA) and genes from the 1918 influenza virus to evaluate the duration of cross-protection to the H1N1 pandemic strain by vaccinating young mice (8 to 12 weeks) and then allowing the animals to age to 20 months. This immunity was long lasting, with homologous receptor-blocking antibodies detected throughout the lifespan of vaccinated mice. Furthermore, the 1918 VLPs fully protected aged mice from 2009 pandemic H1N1 virus challenge 16 months after vaccination. Histopathological assessment showed that aged vaccinated mice had significant protection from alveolar infection but less protection of the bronchial tissue than adult vaccinated mice. Additionally, passive transfer of immune serum from aged vaccinated mice resulted in protection from death but not morbidity. This is the first report describing the lifelong duration of cross-reactive immune responses elicited by a 1918 VLP vaccine in a murine model. Importantly, these lifelong immune responses did not result in decreased total viral replication but did prevent infection of the lower respiratory tract. These findings show that immunity acquired early in life can restrict the anatomical location of influenza viral replication, rather than preventing infection, in the aged.

A computationally optimized hemagglutinin virus-like particle vaccine elicits broadly reactive antibodies that protect nonhuman primates from H5N1 infection.

BACKGROUND: Highly pathogenic H5N1 avian influenza viruses continue to spread via waterfowl, causing lethal infections in humans. Vaccines can prevent the morbidity and mortality associated with pandemic influenza isolates. Predicting the specific isolate that may emerge from the 10 different H5N1 clades is a tremendous challenge for vaccine design. METHODS: In this study, we generated a synthetic hemagglutinin (HA) on the basis of a new method, computationally optimized broadly reactive antigen (COBRA), which uses worldwide sequencing and surveillance efforts that are specifically focused on sequences from H5N1 clade 2 human isolates. RESULTS: Cynomolgus macaques vaccinated with COBRA clade 2 HA H5N1 virus-like particles (VLPs) had hemagglutination-inhibition antibody titers that recognized a broader number of representative isolates from divergent clades as compared to nonhuman primates vaccinated with clade 2.2 HA VLPs. Furthermore, all vaccinated animals were protected from A/Whooper Swan/Mongolia/244/2005 (WS/05) clade 2.2 challenge, with no virus detected in the nasal or tracheal washes. However, COBRA VLP-vaccinated nonhuman primates had reduced lung inflammation and pathologic effects as compared to those that received WS/05 VLP vaccines. CONCLUSIONS: The COBRA clade 2 HA H5N1 VLP elicits broad humoral immunity against multiple H5N1 isolates from different clades. In addition, the COBRA VLP vaccine is more effective than a homologous vaccine against a highly pathogenic avian influenza virus challenge.

Innate immune activation without immune cell infiltration in brains of murine models of Aicardi-Goutières Syndrome.

Chronic inflammation is frequently invoked as a mechanism of neurodegeneration and yet inflammatory cell infiltrates are seldom seen in brains of these disorders. Different disciplines utilize different technologies and methodologies to describe what is immunologically defined as the innate immune response (IIR). We examined murine models of the human neurodegenerative disease Aicardi-Goutières Syndrome, where an IIR is initiated by aberrant RNA metabolism secondary to a mutation in adenosine deaminase acting on RNA gene (ADAR1). We previously showed that these mice demonstrated a deficit in RNA editing that lead to MDA-5 mediated RNA sensing pathway activation of the IIR with massive interferon stimulated gene transcription and translation. As early as 2 weeks of age, in situ hybridization demonstrated that different central nervous system (CNS) cell lineages expressed very high levels of distinct interferon stimulated genes (ISGs) in the absence of interferon and absence of immune cell infiltrates. We have expanded these studies to more completely describe the breadth of ISG expression systemically and in CNS using double label in situ hybridization. Within the CNS aberrant ISG expression was mostly limited to neurons, microglia, ependyma, choroid plexus, and endothelial cells with little expression in oligodendroglia and astrocytes except for STAT1. Wild type controls showed a similar pattern of ISG expression but only in aged mice and at levels minimally detectable by in situ hybridization. Despite months of elevated ISG expression in mutant mice, there was essentially no inflammatory infiltrate, no interferon production and minimal glial reaction. Histomorphological neurodegenerative pathology of ventricular dilatation and deep gray matter mineralization were evident in mutant mice 8-13 months of age but this did not show a spatial relationship to ISG expression. This IIR without immune cell infiltration leads to neurodegeneration through non-canonical pathways that may accentuate normal aging pathways.

Preventive immunization of aged and juvenile non-human primates to ß-amyloid.

BACKGROUND: Immunization against beta-amyloid (Aß) is a promising approach for the treatment of Alzheimer's disease, but the optimal timing for the vaccination remains to be determined. Preventive immunization approaches may be more efficacious and associated with fewer side-effects; however, there is only limited information available from primate models about the effects of preclinical vaccination on brain amyloid composition and the neuroinflammatory milieu. METHODS: Ten non-human primates (NHP) of advanced age (18-26 years) and eight 2-year-old juvenile NHPs were immunized at 0, 2, 6, 10 and 14 weeks with aggregated Aß42 admixed with monophosphoryl lipid A as adjuvant, and monitored for up to 6 months. Anti-Aß antibody levels and immune activation markers were assessed in plasma and cerebrospinal fluid samples before and at several time-points after immunization. Microglial activity was determined by [(11)C]PK11195 PET scans acquired before and after immunization, and by post-mortem immunohistochemical and real-time PCR evaluation. Aß oligomer composition was assessed by immunoblot analysis in the frontal cortex of aged immunized and non-immunized control animals. RESULTS: All juvenile animals developed a strong and sustained serum anti-Aß IgG antibody response, whereas only 80 % of aged animals developed detectable antibodies. The immune response in aged monkeys was more delayed and significantly weaker, and was also more variable between animals. Pre- and post-immunization [(11)C]PK11195 PET scans showed no evidence of vaccine-related microglial activation. Post-mortem brain tissue analysis indicated a low overall amyloid burden, but revealed a significant shift in oligomer size with an increase in the dimer:pentamer ratio in aged immunized animals compared with non-immunized controls (P < 0.01). No differences were seen in microglial density or expression of classical and alternative microglial activation markers between immunized and control animals. CONCLUSIONS: Our results indicate that preventive Aß immunization is a safe therapeutic approach lacking adverse CNS immune system activation or other serious side-effects in both aged and juvenile NHP cohorts. A significant shift in the composition of soluble oligomers towards smaller species might facilitate removal of toxic Aß species from the brain.