IMMUNOCAMOUFLAGE: BROAD-SPECTRUM ANTIVIRAL PROPHYLAXIS in .NET

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IMMUNOCAMOUFLAGE: BROAD-SPECTRUM ANTIVIRAL PROPHYLAXIS
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3 30 minutes. Viral entry and infection was determined by either immunostaining for virus-speci c viral products (SV40, T antigen; CMV, IE72) or by plaque assays. Viral infection was determined by plaque assays (SV40, TMEV, MAV, and RCV) or by intracellular immunostaining for early virus-speci c antigens [T antigen for SV40 and the immediate early 72 kDa protein (IE72) for CMV]. For plaque assays, host cells were grown until 75 90% con uent in 35 mm petri dishes overlaid with 1 mL of the viral stock dilution (10 3 10 8 of viral stocks) for 2 hour incubation at room temperature. Following removal of the remaining viral lysate, the monolayer was overlaid with 4 mL of the plaque overlay media [equal parts 0.6% melted Bacto-Agar and 2 MEM (without phenol red, Gibco) with 10% FBS]. On day 5 to 9 (virus-dependent), the agar was removed from each plate and 1 mL of ice cold methanol was added and incubated for 10 minutes. The methanol was removed and 1 mL of neutral red was added and incubated for 30 minutes. Following removal of the neutral red solution, the plates were dried overnight for visualization of the plaques. Plaques appeared as clear areas where infected cells had died, while living cells stained red.49
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16.3 IMMUNOCAMOUFLAGE: BROAD-SPECTRUM ANTIVIRAL PROPHYLAXIS As hypothesized in Figure 16.1, only covalently grafted mPEG was capable of preventing viral infection (Figure 16.3). Indeed, as demonstrated in Figure 16.3B, the presence of soluble (noncovalently bound) mPEG did not inhibit viral (SV40) entry or propagation over the 72 hour time course as demonstrated by T antigen immunodetection. In contrast, pegylation of either the virus (SV40; Figure 16.3A) or host cell (CV-1; Figure 16.3C) resulted in a dose-dependent decrease in viral infection as determined by T antigen expression. This antiviral prophylaxis was highly effective even at very low (e.g., 0.2 mM) derivatization concentrations. Similar ndings were observed using a rat coronavirus (RCV) model. As shown in Figure 16.4, even very low levels of mPEG grafting (0.2 mM BTCmPEG) to the host cell (L2 rat lung epithelial cells) resulted in an almost complete prevention of viral entry and propagation as determined by plaque assays. Furthermore, at derivatization concentrations !5 mM BTCmPEG, a complete abrogation of viral entry was observed. Photomicrographs further document the effectiveness of mPEG grafting. As shown in Figure 16.4, plaques are readily identi ed in the control infections while the mPEG modi ed L2 monolayers remain uninfected following viral challenge. Importantly, RCV is a member of the same viral family as the SARS virus, thus suggesting that mPEG grafting to nasopharyngeal epithelium may similarly prove to be an effective prophylactic approach. In contrast to existing antiviral prophylactic approaches, mPEG grafting proved surprisingly effective against a broad spectrum of viral families. As shown in Table 16.1 and Figure 16.5, pegylation of the host cells provides potent prophylactic protection against adeno (MAV), picorna (TMEV), papova (SV40), and corona (RCV) viruses. Using a grafting concentration of 5 mM BTCmPEG (5 kDa)
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BROAD-SPECTRUM ANTIVIRAL PROPHYLAXIS: INHIBITION OF VIRAL INFECTION
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FIGURE 16.3 Covalent grafting of BTCmPEG (5 kDa) is essential for antiviral activity. (A) Covalent grafting of mPEG directly to SV40 results in a mPEG dose-dependent decrease in viral entry and T antigen expression at 24, 48, and 72 hours post viral challenge. (B) Large T antigen immunostaining of SV40-infected CV-1 cells 72 hours postchallenge. As noted, nearly 100% of cells in the control infection are T antigen positive while challenge of mPEGgrafted CV-1 cells results in low T antigen expression at 72 hours postchallenge. The presence of high concentrations (15 mM) of soluble mPEG exhibits no antiviral effect. (C) Covalent grafting of mPEG to the host cell prevents SV40 entry and T antigen expression in a mPEG dose-dependent manner. Data presented are the mean SD of a minimum of three independent experiments.
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