Human and murine IFIT1 proteins do not restrict infection of negative-sense RNA viruses of the Orthomyxoviridae, Bunyaviridae, and Filoviridae families

UNLABELLED: Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) is a host protein with reported cell-intrinsic antiviral activity against several RNA viruses. The proposed basis for the activity against negative-sense RNA viruses is the binding to exposed 5\u27-triphosphates (5\u27-ppp) on the genome of viral RNA. However, recent studies reported relatively low binding affinities of IFIT1 for 5\u27-ppp RNA, suggesting that IFIT1 may not interact efficiently with this moiety under physiological conditions. To evaluate the ability of IFIT1 to have an impact on negative-sense RNA viruses, we infected Ifit1(-/-) and wild-type control mice and primary cells with four negative-sense RNA viruses (influenza A virus [IAV], La Crosse virus [LACV], Oropouche virus [OROV], and Ebola virus) corresponding to three distinct families. Unexpectedly, a lack of Ifit1 gene expression did not result in increased infection by any of these viruses in cell culture. Analogously, morbidity, mortality, and viral burdens in tissues were identical between Ifit1(-/-) and control mice after infection with IAV, LACV, or OROV. Finally, deletion of the human IFIT1 protein in A549 cells did not affect IAV replication or infection, and reciprocally, ectopic expression of IFIT1 in HEK293T cells did not inhibit IAV infection. To explain the lack of antiviral activity against IAV, we measured the binding affinity of IFIT1 for RNA oligonucleotides resembling the 5\u27 ends of IAV gene segments. The affinity for 5\u27-ppp RNA was approximately 10-fold lower than that for non-2\u27-O-methylated (cap 0) RNA oligonucleotides. Based on this analysis, we conclude that IFIT1 is not a dominant restriction factor against negative-sense RNA viruses. IMPORTANCE: Negative-sense RNA viruses, including influenza virus and Ebola virus, have been responsible for some of the most deadly outbreaks in recent history. The host interferon response and induction of antiviral genes contribute to the control of infections by these viruses. IFIT1 is highly induced after virus infection and reportedly has antiviral activity against several RNA and DNA viruses. However, its role in restricting infection by negative-sense RNA viruses remains unclear. In this study, we evaluated the ability of IFIT1 to inhibit negative-sense RNA virus replication and pathogenesis both in vitro and in vivo. Detailed cell culture and animal studies demonstrated that IFIT1 is not a dominant restriction factor against three different families of negative-sense RNA viruses

Mouse Models of Heterologous Flavivirus Immunity: A Role for Cross-Reactive T Cells

Most of the world is at risk of being infected with a flavivirus such as dengue virus, West Nile virus, yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, and Zika virus, significantly impacting millions of lives. Importantly, many of these genetically similar viruses co-circulate within the same geographic regions, making it likely for individuals living in areas of high flavivirus endemicity to be infected with multiple flaviviruses during their lifetime. Following a flavivirus infection, a robust virus-specific T cell response is generated and the memory recall of this response has been demonstrated to provide long-lasting immunity, protecting against reinfection with the same pathogen. However, multiple studies have shown that this flavivirus specific T cell response can be cross-reactive and active during heterologous flavivirus infection, leading to the question: How does immunity to one flavivirus shape immunity to the next, and how does this impact disease? It has been proposed that in some cases unfavorable disease outcomes may be caused by lower avidity cross-reactive memory T cells generated during a primary flavivirus infection that preferentially expand during a secondary heterologous infection and function sub optimally against the new pathogen. While in other cases, these cross-reactive cells still have the potential to facilitate cross-protection. In this review, we focus on cross-reactive T cell responses to flaviviruses and the concepts and consequences of T cell cross-reactivity, with particular emphasis linking data generated using murine models to our new understanding of disease outcomes following heterologous flavivirus infection

Protection by immunoglobulin dual-affinity retargeting antibodies against dengue virus

Dengue viruses are the most common arthropod-transmitted viral infection, with an estimated 390 million human infections annually and ∼3.6 billion people at risk. Currently, there are no approved vaccines or therapeutics available to control the global dengue virus disease burden. In this study, we demonstrate the binding, neutralizing activity, and therapeutic capacity of a novel bispecific dual-affinity retargeting molecule (DART) that limits infection of all four serotypes of dengue virus

Defining new therapeutics using a more immunocompetent mouse model of antibody-enhanced dengue virus infection

With over 3.5 billion people at risk and approximately 390 million human infections per year, dengue virus (DENV) disease strains health care resources worldwide. Previously, we and others established models for DENV pathogenesis in mice that completely lack subunits of the receptors (Ifnar and Ifngr) for type I and type II interferon (IFN) signaling; however, the utility of these models is limited by the pleotropic effect of these cytokines on innate and adaptive immune system development and function. Here, we demonstrate that the specific deletion of Ifnar expression on subsets of murine myeloid cells (LysM Cre(+) Ifnar(flox/flox) [denoted as Ifnar(f/f) herein]) resulted in enhanced DENV replication in vivo. The administration of subneutralizing amounts of cross-reactive anti-DENV monoclonal antibodies to LysM Cre(+) Ifnar(f/f) mice prior to infection with DENV serotype 2 or 3 resulted in antibody-dependent enhancement (ADE) of infection with many of the characteristics associated with severe DENV disease in humans, including plasma leakage, hypercytokinemia, liver injury, hemoconcentration, and thrombocytopenia. Notably, the pathogenesis of severe DENV-2 or DENV-3 infection in LysM Cre(+) Ifnar(f/f) mice was blocked by pre- or postexposure administration of a bispecific dual-affinity retargeting molecule (DART) or an optimized RIG-I receptor agonist that stimulates innate immune responses. Our findings establish a more immunocompetent animal model of ADE of infection with multiple DENV serotypes in which disease is inhibited by treatment with broad-spectrum antibody derivatives or innate immune stimulatory agents

Chikungunya virus infection results in higher and persistent viral replication in aged Rhesus macaques due to defects in anti-viral immunity

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne Alphavirus that causes a clinical disease involving fever, myalgia, nausea and rash. The distinguishing feature of CHIKV infection is the severe debilitating poly-arthralgia that may persist for several months after viral clearance. Since its re-emergence in 2004, CHIKV has spread from the Indian Ocean region to new locations including metropolitan Europe, Japan, and even the United States. The risk of importing CHIKV to new areas of the world is increasing due to high levels of viremia in infected individuals as well as the recent adaptation of the virus to the mosquito species Aedes albopictus. CHIKV re-emergence is also associated with new clinical complications including severe morbidity and, for the first time, mortality. In this study, we characterized disease progression and host immune responses in adult and aged Rhesus macaques infected with either the recent CHIKV outbreak strain La Reunion (LR) or the West African strain 37997. Our results indicate that following intravenous infection and regardless of the virus used, Rhesus macaques become viremic between days 1-5 post infection. While adult animals are able to control viral infection, aged animals show persistent virus in the spleen. Virus-specific T cell responses in the aged animals were reduced compared to adult animals and the B cell responses were also delayed and reduced in aged animals. Interestingly, regardless of age, T cell and antibody responses were more robust in animals infected with LR compared to 37997 CHIKV strain. Taken together these data suggest that the reduced immune responses in the aged animals promotes long-term virus persistence in CHIKV-LR infected Rhesus monkeys

Population-level emergence of bedaquiline and clofazimine resistance-associated variants among patients with drug-resistant tuberculosis in southern Africa: a phenotypic and phylogenetic analysis.

BACKGROUND: Bedaquiline and clofazimine are important drugs in the treatment of drug-resistant tuberculosis and are commonly used across southern Africa, although drug susceptibility testing is not routinely performed. In this study, we did a genotypic and phenotypic analysis of drug-resistant Mycobacterium tuberculosis isolates from cohort studies in hospitals in KwaZulu-Natal, South Africa, to identify resistance-associated variants (RAVs) and assess the extent of clofazimine and bedaquiline cross-resistance. We also used a comprehensive dataset of whole-genome sequences to investigate the phylogenetic and geographical distribution of bedaquiline and clofazimine RAVs in southern Africa. METHODS: In this study, we included M tuberculosis isolates reported from the PRAXIS study of patients with drug-resistant tuberculosis treated with bedaquiline (King Dinuzulu Hospital, Durban) and three other cohort studies of drug-resistant tuberculosis in other KwaZulu-Natal hospitals, and sequential isolates from six persistently culture-positive patients with extensively drug-resistant tuberculosis at the KwaZulu-Natal provincial referral laboratory. Samples were collected between 2013 and 2019. Microbiological cultures were done as part of all parent studies. We sequenced whole genomes of included isolates and measured bedaquiline and clofazimine minimum inhibitory concentrations (MICs) for isolates identified as carrying any Rv0678 variant or previously published atpE, pepQ, and Rv1979c RAVs, which were the subject of the phenotypic study. We combined all whole-genome sequences of M tuberculosis obtained in this study with publicly available sequence data from other tuberculosis studies in southern Africa (defined as the countries of the Southern African Development Community), including isolates with Rv0678 variants identified by screening public genomic databases. We used this extended dataset to reconstruct phylogenetic relationships across lineage 2 and 4 M tuberculosis isolates. FINDINGS: We sequenced the whole genome of 648 isolates from 385 patients with drug-resistant tuberculosis recruited into cohort studies in KwaZulu-Natal, and 28 isolates from six patients from the KwaZulu-Natal referral laboratory. We identified 30 isolates with Rv0678 RAVs from 16 (4%) of 391 patients. We did not identify any atpE, pepQ, or Rv1979c RAVs. MICs were measured for 21 isolates with Rv0678 RAVs. MICs were above the critical concentration for bedaquiline resistance in nine (43%) of 21 isolates, in the intermediate category in nine (43%) isolates, and within the wild-type range in three (14%) isolates. Clofazimine MICs in genetically wild-type isolates ranged from 0·12-0·5 μg/mL, and in isolates with RAVs from 0·25-4·0 μg/mL. Phylogenetic analysis of the extended dataset including M tuberculosis isolates from southern Africa resolved multiple emergences of Rv0678 variants in lineages 2 and 4, documented two likely nosocomial transmission events, and identified the spread of a possibly bedaquiline and clofazimine cross-resistant clone in eSwatini. We also identified four patients with pepQ frameshift mutations that may confer resistance. INTERPRETATION: Bedaquiline and clofazimine cross-resistance in southern Africa is emerging repeatedly, with evidence of onward transmission largely due to Rv0678 mutations in M tuberculosis. Roll-out of bedaquiline and clofazimine treatment in the setting of limited drug susceptibility testing could allow further spread of resistance. Designing strong regimens would help reduce the emergence of resistance. Drug susceptibility testing is required to identify where resistance does emerge. FUNDING: Wellcome Trust, National Institute of Allergy and Infectious Diseases and National Center for Advancing Translational Sciences of the National Institutes of Health

Key role of T cell defects in age-related vulnerability to West Nile virus

West Nile virus (WNV) infection causes a life-threatening meningoencephalitis that becomes increasingly more prevalent over the age of 50 and is 40–50× more prevalent in people over the age of 70, compared with adults under the age of 40. In a mouse model of age-related vulnerability to WNV, we demonstrate that death correlates with increased viral titers in the brain and that this loss of virus control with age was the result of defects in the CD4 and CD8 T cell response against WNV. Specific age-related defects in T cell responses against dominant WNV epitopes were detected at the level of cytokine and lytic granule production, each of which are essential for resistance against WNV, and in the ability to generate multifunctional anti-WNV effector T cells, which are believed to be critical for robust antiviral immunity. In contrast, at the peak of the response, old and adult T cells exhibited superimposable peptide sensitivity. Most importantly, although the adult CD4 or CD8 T cells readily protected immunodeficient mice upon adoptive transfer, old T cells of either subset were unable to provide WNV-specific protection. Consistent with a profound qualitative and quantitative defect in T cell immunity, old brains contained at least 12× fewer total effector CD8 T cells compared with adult mice at the peak of brain infection. These findings identify potential targets for immunomodulation and treatment to combat lethal WNV infection in the elderly

Functional analysis of antibodies against dengue virus type 4 reveals strain-dependent epitope exposure that impacts neutralization and protection

Although prior studies have characterized the neutralizing activities of monoclonal antibodies (MAbs) against dengue virus (DENV) serotypes 1, 2, and 3 (DENV-1, DENV-2, and DENV-3), few reports have assessed the activity of MAbs against DENV-4. Here, we evaluated the inhibitory activity of 81 new mouse anti-DENV-4 MAbs. We observed strain- and genotype-dependent differences in neutralization of DENV-4 by MAbs mapping to epitopes on domain II (DII) and DIII of the envelope (E) protein. Several anti-DENV-4 MAbs inefficiently inhibited at least one strain and/or genotype, suggesting that the exposure or sequence of neutralizing epitopes varies within isolates of this serotype. Remarkably, flavivirus cross-reactive MAbs, which bound to the highly conserved fusion loop in DII and inhibited infection of DENV-1, DENV-2, and DENV-3, more weakly neutralized five different DENV-4 strains encompassing the genetic diversity of the serotype after preincubation at 37°C. However, increasing the time of preincubation at 37°C or raising the temperature to 40°C enhanced the potency of DII fusion loop-specific MAbs and some DIII-specific MAbs against DENV-4 strains. Prophylaxis studies in two new DENV-4 mouse models showed that neutralization titers of MAbs after preincubation at 37°C correlated with activity in vivo. Our studies establish the complexity of MAb recognition against DENV-4 and suggest that differences in epitope exposure relative to other DENV serotypes affect antibody neutralization and protective activity

Zika virus pathogenesis in rhesus macaques is unaffected by pre-existing immunity to dengue virus

Zika virus (ZIKV) is a re-emerging virus that has recently spread into dengue virus (DENV) endemic regions and cross-reactive antibodies (Abs) could potentially affect ZIKV pathogenesis. Using DENV-immune serum, it has been shown in vitro that antibody-dependent enhancement (ADE) of ZIKV infection can occur. Here we study the effects of pre-existing DENV immunity on ZIKV infection in vivo. We infect two cohorts of rhesus macaques with ZIKV; one cohort has been exposed to DENV 2.8 years earlier and a second control cohort is naïve to flaviviral infection. Our results, while confirming ADE in vitro, suggest that pre-existing DENV immunity does not result in more severe ZIKV disease. Rather our results show a reduction in the number of days of ZIKV viremia compared to naïve macaques and that the previous exposure to DENV may result in modulation of the immune response without resulting in enhancement of ZIKV pathogenesis