Rebecca Broeckel

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.

Characterization of a Novel Human-Specific STING Agonist that Elicits Antiviral Activity Against Emerging Alphaviruses

Pharmacologic stimulation of innate immune processes represents an attractive strategy to achieve multiple therapeutic outcomes including inhibition of virus replication, boosting antitumor immunity, and enhancing vaccine immunogenicity. In light of this we sought to identify small molecules capable of activating the type I interferon (IFN) response by way of the transcription factor IFN regulatory factor 3 (IRF3). A high throughput in vitro screen yielded 4-(2-chloro-6-fluorobenzyl)-N-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide (referred to herein as G10), which was found to trigger IRF3/IFN-associated transcription in human fibroblasts. Further examination of the cellular response to this molecule revealed expression of multiple IRF3-dependent antiviral effector genes as well as type I and III IFN subtypes. This led to the establishment of a cellular state that prevented replication of emerging Alphavirus species including Chikungunya virus, Venezuelan Equine Encephalitis virus, and Sindbis virus. To define cellular proteins essential to elicitation of the antiviral activity by the compound we employed a reverse genetics approach that utilized genome editing via CRISPR/Cas9 technology. This allowed the identification of IRF3, the IRF3-activating adaptor molecule STING, and the IFN-associated transcription factor STAT1 as required for observed gene induction and antiviral effects. Biochemical analysis indicates that G10 does not bind to STING directly, however. Thus the compound may represent the first synthetic small molecule characterized as an indirect activator of human STING-dependent phenotypes. In vivo stimulation of STING-dependent activity by an unrelated small molecule in a mouse model of Chikungunya virus infection blocked viremia demonstrating that pharmacologic activation of this signaling pathway may represent a feasible strategy for combating emerging Alphaviruses.

Zika virus infection in pregnant rhesus macaques causes placental dysfunction and immunopathology

Zika virus (ZIKV) infection during pregnancy leads to an increased risk of fetal growth restriction and fetal central nervous system malformations, which are outcomes broadly referred to as the Congenital Zika Syndrome (CZS). Here we infect pregnant rhesus macaques and investigate the impact of persistent ZIKV infection on uteroplacental pathology, blood flow, and fetal growth and development. Despite seemingly normal fetal growth and persistent fetal-placenta-maternal infection, advanced non-invasive in vivo imaging studies reveal dramatic effects on placental oxygen reserve accompanied by significantly decreased oxygen permeability of the placental villi. The observation of abnormal oxygen transport within the placenta appears to be a consequence of uterine vasculitis and placental villous damage in ZIKV cases. In addition, we demonstrate a robust maternal-placental-fetal inflammatory response following ZIKV infection. This animal model reveals a potential relationship between ZIKV infection and uteroplacental pathology that appears to affect oxygen delivery to the fetus during development.Zika virus infection during pregnancy can result in birth defects, but underlying pathogenesis at the maternal-fetal interface is unclear. Here, the authors use non-invasive in vivo imaging of Zika-infected rhesus macaques and show that infection results in abnormal oxygen transport across the placenta.

Nonhuman Primate Models of Chikungunya Virus Infection and Disease (CHIKV NHP Model)

Chikungunya virus (CHIKV) is a positive-sense RNA virus transmitted by Aedes mosquitoes. CHIKV is a reemerging Alphavirus that causes acute febrile illness and severe and debilitating polyarthralgia of the peripheral joints. Huge epidemics and the rapid spread of CHIKV seen in India and the Indian Ocean region established CHIKV as a global health concern. This concern was further solidified by the recent incursion of the virus into the Western hemisphere, a region without pre-existing immunity. Nonhuman primates (NHPs) serve as excellent animal models for understanding CHIKV pathogenesis and pre-clinical assessment of vaccines and therapeutics. NHPs present advantages over rodent models because they are a natural amplification host for CHIKV and they share significant genetic and physiological homology with humans. CHIKV infection in NHPs results in acute fever, rash, viremia and production of type I interferon. NHPs develop CHIKV-specific B and T-cells, generating neutralizing antibodies and CHIKV-specific CD4+ and CD8+ T-cells. CHIKV establishes a persistent infection in NHPs, particularly in cynomolgus macaques, because infectious virus could be recovered from spleen, liver, and muscle as late as 44 days post infection. NHPs are valuable models that are useful in preclinical testing of vaccines and therapeutics and uncovering the details of CHIKV pathogenesis.

Dysregulated TGF-β Production Underlies the Age-Related Vulnerability to Chikungunya Virus

Chikungunya virus (CHIKV) is a re-emerging global pathogen with pandemic potential, which causes fever, rash and debilitating arthralgia. Older adults over 65 years are particularly susceptible to severe and chronic CHIKV disease (CHIKVD), accounting for >90% of all CHIKV-related deaths. There are currently no approved vaccines or antiviral treatments available to limit chronic CHIKVD. Here we show that in old mice excessive, dysregulated TGFβ production during acute infection leads to a reduced immune response and subsequent chronic disease. Humans suffering from CHIKV infection also exhibited high TGFβ levels and a pronounced age-related defect in neutralizing anti-CHIKV antibody production. In vivo reduction of TGFβ levels minimized acute joint swelling, restored neutralizing antibody production and diminished chronic joint pathology in old mice. This study identifies increased and dysregulated TGFβ secretion as one key mechanism contributing to the age-related loss of protective anti-CHIKV-immunity leading to chronic CHIKVD.

Additives for vaccine storage to improve thermal stability of adenoviruses from hours to months

Up to 80% of the cost of vaccination programmes is due to the cold chain problem (that is, keeping vaccines cold). Inexpensive, biocompatible additives to slow down the degradation of virus particles would address the problem. Here we propose and characterize additives that, already at very low concentrations, improve the storage time of adenovirus type 5. Anionic gold nanoparticles (10−8–10−6 M) or polyethylene glycol (PEG, molecular weight ∼8,000 Da, 10−7–10−4 M) increase the half-life of a green fluorescent protein expressing adenovirus from ∼48 h to 21 days at 37 °C (from 7 to >30 days at room temperature). They replicate the known stabilizing effect of sucrose, but at several orders of magnitude lower concentrations. PEG and sucrose maintained immunogenicity in vivo for viruses stored for 10 days at 37 °C. To achieve rational design of viral-vaccine stabilizers, our approach is aided by simplified quantitative models based on a single rate-limiting step.

Therapeutic administration of a recombinant human monoclonal antibody reduces the severity of chikungunya virus disease in rhesus macaques

Chikungunya virus (CHIKV) is a mosquito-borne virus that causes a febrile syndrome in humans associated with acute and chronic debilitating joint and muscle pain. Currently no licensed vaccines or therapeutics are available to prevent or treat CHIKV infections. We recently isolated a panel of potently neutralizing human monoclonal antibodies (mAbs), one (4N12) of which exhibited prophylactic and post-exposure therapeutic activity against CHIKV in immunocompromised mice. Here, we describe the development of an engineered CHIKV mAb, designated SVIR001, that has similar antigen binding and neutralization profiles to its parent, 4N12. Because therapeutic administration of SVIR001 in immunocompetent mice significantly reduced viral load in joint tissues, we evaluated its efficacy in a rhesus macaque model of CHIKV infection. Rhesus macaques that were treated after infection with SVIR001 showed rapid elimination of viremia and less severe joint infiltration and disease compared to animals treated with SVIR002, an isotype control mAb. SVIR001 reduced viral burden at the site of infection and at distant sites and also diminished the numbers of activated innate immune cells and levels of pro-inflammatory cytokines and chemokines. SVIR001 therapy; however, did not substantively reduce the induction of CHIKV-specific B or T cell responses. Collectively, these results show promising therapeutic activity of a human anti-CHIKV mAb in rhesus macaques and provide proof-of-principle for its possible use in humans to treat active CHIKV infections.

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

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