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BackgroundAtovaquone, a hydroxynaphthoquinone, FDA Pregnancy category C, used for the treatment and prevention of pneumocysits jirovecii pneumonia (PCP), toxoplasmosis, babesiosis and malaria has in vitro activity against Zika virus (ZIKV). The mechanism of action against Plasmodium spp. and other parasites is based in the inhibition of mitochondrial cytochrome bc1 complex which further collapses parasite–mitochondrial membrane potential. But to date, antiviral activity of this drug has not been described.MethodsVero cells (monkey kidney epithelial cells) were seeded. At 24 hours of incubation, the cells were pretreated with ribavirin and brequinar (known antiviral drugs) and atovaquone at different concentrations for 1 hour and then infected with ZIKV Brazilian strain and Ugandan strain, and subsequently treated with drugs again. After incubation for 72 hours virus antigen Env-protein production was quantified by immunodetection. The concentration of atovaquone that decreased the level of Env-protein production by 50% was calculated by non-linear regression analysis (CC50). Cell viability was measured using the CellTiter 96 aqueous one solution cell proliferation assay (Promega, Madison, WI), according to the manufacturers protocol. Viral infection was rescued adding uracil to Vero cells pre-treated with ribavirin, brequinar and atovaquone. Experiment was repeated with Chikungunya virus (CHIKV).ResultsWe found that atovaquone inhibits ZIKV infection in Vero cells at smaller concentration (CC50 = 0.52 μM) than those used for parasitic killing. The effect is more prominent in the Brazilian strain when compared with the Ugandan strain. No cytotoxic effect was found in Vero cells up to 15 μM; above this concentration atovaquone formed crystals. Uracil rescues ZIKV infection after treatment with atovaquone. Atovaquone also inhibited CHIKV infection in Vero cells. ConclusionAtovaquone has antiviral activity against ZIKV likely via depletion of nucleotides blocking pyrimidine biosynthesis. Furthermore, the antiviral effect is applicable to other arboviruses which makes atovaquone a broad-spectrum antiviral drug and a potential attractive candidate for the treatment of ZIKV infection in vulnerable population such pregnant women and children.Disclosures All authors: No reported disclosures.

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Interleukin-22 (IL-22), an IL-10 cytokine family member, is produced by a variety of immune cells including Th1, Th17 and Th22 T helper cells, CD8+ T-cells, NK cells, ILC3, neutrophils and macrophages. Interleukin-22 crucially participates in regulating the integrity of epithelial barriers by modulating inflammation, mucus production, additional aspects of wound healing and has been noted to be substantially induced in patients with various chronic inflammatory conditions. Upregulation of IL-22 in psoriasis (PS), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) inflammatory bowel disease, Crohn’s disease, cystic fibrosis and atopic dermatitis (AD) has been well documented, with IL-22 often considered a hallmark of IL-1-driven immune responses. Furthermore, IL-22 protein has been proposed as a stratification biomarker for psoriatic arthritis therapeutic choice between IL-17 and TNF inhibitions, to correlate with the severity of clinical sequelae of Sjogren’s Syndrome, and IL-22 mRNA expression stratifies AD responses to fezakinumab. In healthy individuals, IL-22 activity resulting from low level expression may be functionally neutralized, at least in selected tissues such as the intestinal tract, by binding to soluble IL-22 binding protein (IL-22BP) expressed locally by dendritic cells. However, in these pathologies, inhibition of IL-22 by IL-22BP is presumably overcome by elevated expression of IL-22 protein. Explore ranges of IL-22 serum concentrations to differentiate between healthy donors (HD) and select autoimmune disease patients with a simple-to-use, high sensitivity ELISA. A commercial sandwich ELISA (PBL Assay Science, 41701-1) quantifying human IL-22 was characterized and used to assess IL-22 concentration in sera from HD and patients exhibiting one of several autoimmune diseases. This high sensitivity ELISA, having a manufacturer-stated LLOQ of 0.78 pg/ml, exhibited good assay precision, dilutional linearity and spike recovery using HD serum. Parallelism was demonstrated using serum samples from four patients with AD and three HD. Addition of a 10- to 100-fold mass excess of exogenous recombinant IL-22BP reduced the ability of the assay to quantify IL-22 suggesting that at least one antigenic region recognized by ELISA antibodies may be sterically hindered by IL-22BP binding to IL-22. However, such high levels of 10 and 100 ng/ml of IL-22BP are unlikely to be achieved in vivo, and therefore may not adversely affect quantitation of physiological levels of IL-22 protein by this assay. Commercially sourced HD sera concentrations of IL-22 exceeded the assay LLOQ in 23 of 24 samples (96%) with a mean IL-22 concentration of 1.84 ± 1.33 pg/ml. Commercial sera from patients with AD (n = 10, 100%), PS (n = 10, 100%), RA (n = 10, 100%), SLE (n = 10, 100%) were similarly analysed and yielded mean levels of 14.8 ± 11.2, 5.1 ± 2.4, 5.6 ± 0.91 and 3.1 ± 0.74 pg/mL, respectively. Interleukin-22 measured in AD (and PS) sera exhibited sufficiently broad ranges of concentrations such that statistical differences between AD and all other groups were not readily achievable with this number of samples. Notably, these commercially obtained serum samples were not controlled for overall severity of any disease at the time of sample collection. Sample sets were randomly obtained from a population of individuals self-identifying as AD sufferers, as were the other patient samples. With mean serum IL-22 in AD appearing to trend as substantially elevated, an opportunity exists to explore IL-22 trends along with other biomarkers in clinically identified and possibly symptom-stratified populations of patients with AD. Should this trend toward IL-22 elevation be fully substantiated in AD, therapeutic stratification and precision medicine approaches may be conceivable based on serum IL-22 protein concentrations, supporting other investigators’ earlier work examining IL-22 mRNA. The baseline readability of endogenous IL-22 in HD sera using this ELISA is also highly advantageous.

We examined Interferon (IFN)-Alpha and -Beta levels in pooled sera and individual bleeds from C57/Bl, Balb/C and CD-1 mice. 20 Individuals from each strain from two independent colonies were evaluated in commercial assays. The IFN-Beta ELISA detected IFN in 1 of the 60 individuals while the IFN-Alpha assay which detects all 14 subtypes measured IFN in 13 of 60. 5 individuals exhibited greater than 4 pg/ml of IFN-Alpha detected. Specificity was demonstrated by knockdown of the signal using non-biotinylated detection antibody. 14 of 54 porcine sera samples from 4 independent sources were positive for IFN alpha by an ELISA under development, and similar knock-down experiments suggest specificity. This is in contrast to human sera/plasma samples where less than 3% of healthy donor samples have quantifiable IFN-Alpha or -Beta above 0.5 pg/ml. This study may raise questions about the regulation of the IFN system in different species and the potential of these differences to affect experimental results in a variety of models.