Development of a new class of antivirals active against pox and measles viruses

In this PhD project we show for the first time that novel dideoxy bicyclic pyrimidine nucleoside analogues (ddBCNAs) with L-chirality represent promising antiviral candidates for use against pox and measles viruses. We suggest a mechanism of action based on a cellular target. Our lead compound (Cf2642, with side chain C9H18–O–C5H11) is active against vaccinia virus (a surrogate poxvirus for smallpox) and measles virus, with IC50 concentrations of 0.19 and 7.5 µM, respectively. This is a 60-fold enhancement over cidofovir (viral DNA polymerase inhibitor; IC50 of 11.5 µM against VACV). A structure activity relationship was established, which was similar for both viruses, indicating a common and specific mechanism of action. Cf2642 does not inhibit HSV-1/2, influenza, adeno or yellow fever viruses. The mechanism of action for the ddBCNAs has been investigated and, though not defined, has been narrowed down. Based on our observations of drug activity in cell lines derived from various sources, we have suggested a cellular target for the ddBCNAs, most likely cellular membrane compartments or the proteins located therein. Though inhibition of vaccinia is observed within two hours of infection, we have shown that the ddBCNAs are unlikely to be entry inhibitors. Acidification of the extracellular medium was observed but, whilst it may be linked to the mechanism of action, this is not the cause of the antiviral effects. With a possible cellular target, toxicity was carefully evaluated. We have not observed significant cytotoxicity in any of our cell models. Antivirals active against cellular targets are less subject to viral resistance, which may develop rapidly with virus-targeting drugs. This could be critical since, there are currently no effective measles antiviral drugs available on the market, and resistance to measles RNA polymerase inhibitors and the potential antipoxviral drug cidofovir has already been described

Identification of Broad-Spectrum Antiviral Compounds by Targeting Viral Entry.

Viruses are a major threat to human health and economic well-being. In recent years Ebola, Zika, influenza, and chikungunya virus epidemics have raised awareness that infections can spread rapidly before vaccines or specific antagonists can be made available. Broad-spectrum antivirals are drugs with the potential to inhibit infection by viruses from different groups or families, which may be deployed during outbreaks when specific diagnostics, vaccines or directly acting antivirals are not available. While pathogen-directed approaches are generally effective against a few closely related viruses, targeting cellular pathways used by multiple viral agents can have broad-spectrum efficacy. Virus entry, particularly clathrin-mediated endocytosis, constitutes an attractive target as it is used by many viruses. Using a phenotypic screening strategy where the inhibitory activity of small molecules was sequentially tested against different viruses, we identified 12 compounds with broad-spectrum activity, and found a subset blocking viral internalisation and/or fusion. Importantly, we show that compounds identified with this approach can reduce viral replication in a mouse model of Zika infection. This work provides proof of concept that it is possible to identify broad-spectrum inhibitors by iterative phenotypic screenings, and that inhibition of host-pathways critical for viral life cycles can be an effective antiviral strategy

Novel antiviral activity of l-dideoxy bicyclic nucleoside analogues versus vaccinia and measles viruses in vitro

Dideoxy bicyclic pyrimidine nucleoside analogues (ddBCNAs) with d-chirality have previously been described by us to inhibit replication of human cytomegalovirus. We herein report for the first time that activity against vaccinia virus (VACV) was achieved using novel l-analogues. A structure–activity relationship was established: Antiviral activity versus VACV was highest with an ether side chain with an optimum of n-C9H18–O–n-C5H11. This gave an IC50 of 190 nM, a 60-fold enhancement over the FDA-approved antiviral cidofovir. Interestingly, l-ddBCNAs also inhibit wild type measles virus syncytia formation with a TCID50 of 7.5 μM for the lead compound. We propose that l-ddBCNAs represent significant innovative antiviral candidates versus measles and poxviruses, and we suggest a mechanism of action versus one or more cellular targets that are essential for viral replication

Seroprevalence of Molluscum contagiosum Virus in German and UK Populations

<div><p>Molluscum contagiosum virus (MCV) is a significant but underreported skin pathogen for children and adults. Seroprevalence studies can help establish burden of disease. Enzyme linked immunosorbent assay (ELISA) based studies have been published for Australian and Japanese populations and the results indicate seroprevalences between 6 and 22 percent in healthy individuals, respectively. To investigate seroprevalence in Europe, we have developed a recombinant ELISA using a truncated MCV virion surface protein MC084 (V123-R230) expressed in <i>E. coli.</i> The ELISA was found to be sensitive and specific, with low inter- and intra-assay variability. Sera from 289 German adults and children aged 0–40 years (median age 21 years) were analysed for antibodies against MC084 by direct binding ELISA. The overall seropositivity rate was found to be 14.8%. The seropositivity rate was low in children below the age of one (4.5%), peaked in children aged 2–10 years (25%), and fell again in older populations (11–40 years; 12.5%). Ten out of 33 healthy UK individuals (30.3%; median age 27 years) had detectable MC084 antibodies. MCV seroconversion was more common in dermatological and autoimmune disorders, than in immunocompromised patients or in patients with multiple sclerosis. Overall MCV seroprevalence is 2.1 fold higher in females than in males in a UK serum collection. German seroprevalences determined in the MC084 ELISA (14.8%) are at least three times higher than incidence of MC in a comparable Swiss population (4.9%). While results are not strictly comparable, this is lower than Australian seroprevalence in a virion based ELISA (n = 357; 23%; 1999), but higher than the seroprevalence reported in a Japanese study using an N-terminal truncation of MC133 (n = 108, 6%; 2000. We report the first large scale serological survey of MC in Europe (n = 393) and the first MCV ELISA based on viral antigen expressed in <i>E. coli.</i></p></div

HaCaT Immunofluorescence.

<p>(A) HaCaT cell culture infected with recombinant vaccinia virus expressing MC084S (v319). Reactivity of high titre human serum HDV0901071 and secondary antibody AlexaFluor 488 (Green) goat anti-human IgG (H+L). (B) HaCaT cell culture infected with recombinant vaccinia virus expressing MC084S (v319). Reactivity of low titre human serum HDV0900040 and secondary antibody AlexaFluor 488 (Green) goat anti-human IgG (H+L). (C) Mock infected cells. Reactivity of high titre human serum HDV0901071 and secondary antibody AlexaFluor 488 (Green) goat anti-human IgG (H+L). Nuclei are stained with DAPI (Hoechst) and shown in blue. Samples were analysed for fluorescence emission properties by using confocal scanning laser microscopy Leica TCS SP2 AOBS.</p

Summary of seroprevalences in German and UK populations.

<p>*SLE – Systemic Lupus Erythematosus.</p>†<p>Autoimm. – General autoimmune conditions.</p>#<p>PPMS – Primary progressive multiple sclerosis.</p><p>^{<>\scale 80%\raster="rg1"\<>}RRMS – Relapsing remitting multiple sclerosis.</p

Bioinformatics.

<p>(A) Transmembrane plot (TMHMM Server v.2.0) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088734#pone.0088734-Krogh1" target="_blank">[25]</a> of mc084 amino acids 1–318; (B) hydropathy plot of MC084 protein with predicted high hydrophilic/antigenic regions indicated by black boxes. The full length ORF (MC084 1–318; predicted molecular weight 34.2 kD; shown on top) was cloned into vRB12 using specific primers tailed with restriction enzyme sites <i>BamHI</i>-<i>HindIII</i>) and C-terminal StrepII epitope tag. The resulting plasmid p319 was sequenced and the recombinant vaccinia virus v319 isolated on BSC-1 cells using the plaqueless mutant system <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088734#pone.0088734-Blasco1" target="_blank">[26]</a>. N- and C-terminal (in yellow) truncations were subcloned from the original full length MCV gene into pGEX-2TK for overexpression in <i>E. coli</i> BL21 (RIL⁺). TMHMM was used to determine transmembrane regions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088734#pone.0088734-Blasco1" target="_blank">[26]</a> whereas the Kyte-Doolittle plot was used to identify hydrophilic regions with predicted high antigenicity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088734#pone.0088734-Kyte1" target="_blank">[27]</a>.</p

Tissue stain details.

<p>Microscopy (4×) of a Molluscum contagiosum lesion section (17315/11) stained with MC patient positive serum (CF2012-1) and haematoxylin-eosin counterstain (upper left hand corner). Three insets showing details at various magnifications [inset 1-(10×), inset 2-(20×) and inset 3-(20×)].</p

Sensitivity.

<p>Absorbance plot of twelve sera from patients clinically diagnosed with MCV (India n = 10; UK n = 2; control group of 0–1 year old individuals n = 17).</p

German seroprevalence.

<p>Distribution of anti-MC084S antibodies in a German population tested by direct binding ELISA (A) Serological responses to MCV antigen MC084 in a German population (n = 289; ages 0–40 years) expressed as the δODU value of an individual serum sample. The horizontal bar within each group represents the median absorbance measurement. (B) Percent seropositivities in different age groups after cut–off of 0.36 (i) 0–1 years (4.5%), (ii) 2–5 years (25%), (iii) 6–10 years (23.4%), 11–20 years (12.5%), and 21–40 years (13.5%).</p