Figure 1.
Ribbon diagrams of the three dimensional structures of the lectins griffithsin and scytovirin.
(A) griffithsin (GRFT), a 25 kDa-protein (a domain-swapped homodimmer of 12.7-kDa subunit) was originally isolated from the red algae Griffithsia sp.; (B) Scytovirin, a 9.7-kDa protein, was originally isolated from the blue-green algae Scytonema varium.
Figure 2.
GRFT and SVN are only weakly active in HCV replicon assay.
GRFT and SVN were tested in the HCV replicon assay, which does not include viral entry targets. HCV replicon assay was done as described in Materials and Methods using either genotype 1b (A) or 2a (B). The y-axis indicates % inhibition of luciferease activity (light units) (diamond) and cell viability (WST-8 assay) (triangle), respectively. Interferon-a (IFN-α) was used as a positive control.
Figure 3.
Comparison of anti-HCV activities of GRFT, CV-N and SVN against JFH-1.
Anti-HCV activities were evaluated by JFH-1 HCVcc full-replication assay, by measuring HCV core protein output in the culture supernatants of Huh7.5.1 cells 72 hr post-infection. The cytotoxicity was evaluated in parallel using WST-8 assay. The HCV output (open circle) and cell viability (closed square) were plotted as percentage (%) relative to control infections without CV-N (A), SVN (B) or GRFT (C). Compounds were added 15 min prior to viral challenge. Results are expressed as mean and standard deviation from triplicate experiments.
Table 1.
Activity of oligomannose-specific lectins from natural product extracts in HCV cell culture assay for inhibition of the type 2a JFH-1 strain of HCV.a
Table 2.
Inhibitory effect of scytovirin and griffithsin on the replication of JFH-1 and HCVcc chimera.a
Figure 4.
GRFT and SVN inhibit HCVpp entry for different genotypes.
Huh7.5.1 cells were incubated for 3 hr with either HCV pseudoparticles(pp) generated with envelope glycoproteins of genotype 2a (JFH-1 and J6 strains) and genotype 1b (TH strain) or VSV-G pp in the presence of increasing amount of GRFT (A) or SVN (B). The inoculum was then removed, and the cells were further incubated. At 2 days post-inoculation, cells were lysed and processed to measure the luciferase activity. The results are presented as percentage of the infectivity relative to infectivity of HCVpp in the absence of carbohydrate binding proteins. Pseudotyped particles produced in the absence of envelope proteins were used as controls, representing <2% of the activity measured for HCVpp. The results are expressed as the mean ± SD in triplicate experiments.
Figure 5.
GRFT and SVN bind to HCV E2 glycoprotein.
Recombinant glycosylated HCV envelope glycoprotein E2 (strain H77 1a) was bound to 96-well plates and exposed to varying concentrations of both SVN and GRFT. The presence of SVN (A) and GRFT (B) was visualized using rabbit polyclonal anti-GRFT or anti-SVN antibodies as described in the Materials and Methods section. Data points represent the mean of three experiments ± SD.
Figure 6.
Plasma levels of GRFT following subcutaneous injection into mice.
GRFT (20 mg/kg day, Q24) was injected into Alb-uPA/SCID transgenic mice for ten consecutive days. Blood was drawn at the indicated days and plasma was measured for GRFT content by ELISA capture assay as described in the Materials and Methods section. Individual animals are shown as red squares (GRFT-treated, n = 5) or black diamonds (control animals, n = 5). Horizontal bold lines represent the mean values for each group.
Figure 7.
GRFT activity in vivo at reducing viral titers after challenge with HCV.
Serum HCV titer changes for saline control (black, n = 6) and griffithsin treated (red, n = 6) Alb-uPA/SCID transgenic mice engrafted with human hepatocytes during a 10-dvday treatment regimen. Error bars show the standard error for values at each time point. The minimum detectable limit of the virus load assay is 200 IU/ml.
Figure 8.
GRFT in vivo activity at reducing viral titers prior to challenge with HCV.
Serum HCV titer changes for griffithsin treated (blue, n = 6), IFN-α treated (red, n = 6), GRFT+IFNα-treated (green, n = 7) and saline control treated (black, n = 6) Alb-uPA/SCID transgenic mice engrafted with human hepatocytes during an 18-day treatment regimen. On Day 31 the differences in mean log titers between the saline control and both single agent treatment groups were statistically significant: saline vs Interferon p = 0.010; saline vs Griffithsin p = 0.020. The difference in mean log titer between the saline and combination groups was not significant (p = 0.053). Error bars show the standard error for values at each time point. The minimum detectable limit of the virus load assay is 200 IU.