Studies in a Murine Model Confirm the Safety of Griffithsin and Advocate Its Further Development as a Microbicide Targeting HIV-1 and Other Enveloped Viruses

Kouokam, Joseph Calvin; Lasnik, Amanda B.; Palmer, Kenneth E.

Next, we assessed the effect of GRFT on mPBMC size and granularity. When cultured in the presence of 1 and 4 µM GRFT, cells showed no difference by flow cytometry analysis compared to PBS treated cells, whereas cells treated with ConA and PHA showed sub-populations of cells with higher forward scatter (FSC) and/or increased side scatter (SSC) values, likely representing activated and enlarged PBMCs. This resulted in a reduction of typical PBMC number as gated in Figure 2A -E and quantified in Figure 2F . These observations were made even without the use of any fluorophore. Likewise, when the cells were loaded with propidium iodide (PI) which discriminates live from dead cells, flow cytometry histograms of mPBMCs treated with GRFT (1 or 4 µM) were similar to that obtained for PBS treated cells ( Figure S1 ). PHA and ConA induced a pronounced cytotoxicity reflected by a shift in histograms (increased PI staining) in comparison with PBS-or GRFT-treated cells ( Figure S1 ). Next, we assessed the effect of GRFT on mPBMC size and granularity. When cultured in the presence of 1 and 4 μM GRFT, cells showed no difference by flow cytometry analysis compared to PBS treated cells, whereas cells treated with ConA and PHA showed sub-populations of cells with higher forward scatter (FSC) and/or increased side scatter (SSC) values, likely representing activated and enlarged PBMCs. This resulted in a reduction of typical PBMC number as gated in Figure 2A -E and quantified in Figure 2F . These observations were made even without the use of any fluorophore. Likewise, when the cells were loaded with propidium iodide (PI) which discriminates live from dead cells, flow cytometry histograms of mPBMCs treated with GRFT (1 or 4 μM) were similar to that obtained for PBS treated cells ( Figure S1 ). PHA and ConA induced a pronounced cytotoxicity reflected by a shift in histograms (increased PI staining) in comparison with PBS-or GRFT-treated cells ( Figure S1 ). Next, we assessed the effect of GRFT on mPBMC size and granularity. When cultured in the presence of 1 and 4 μM GRFT, cells showed no difference by flow cytometry analysis compared to PBS treated cells, whereas cells treated with ConA and PHA showed sub-populations of cells with higher forward scatter (FSC) and/or increased side scatter (SSC) values, likely representing activated and enlarged PBMCs. This resulted in a reduction of typical PBMC number as gated in Figure 2A -E and quantified in Figure 2F . These observations were made even without the use of any fluorophore. Likewise, when the cells were loaded with propidium iodide (PI) which discriminates live from dead cells, flow cytometry histograms of mPBMCs treated with GRFT (1 or 4 μM) were similar to that obtained for PBS treated cells ( Figure S1 ). PHA and ConA induced a pronounced cytotoxicity reflected by a shift in histograms (increased PI staining) in comparison with PBS-or GRFT-treated cells ( Figure S1 ). Furthermore, we evaluated the percentages of cells expressing two known PBMC cellular activation markers, CD25 and CD69. In PBS-treated cells, 3.0% ± 0.2% of cells were CD4+CD25+ ( Figure 3A ). mPBMCs incubated in the presence of 1 and 4 µM GRFT showed similar values for CD4+/CD25+ cells with 3.7% ± 0.23% and 3.8% ± 0.4%, respectively, whereas these amounts were markedly increased by 87 nM, i.e., 10 µg/mL PHA (11.0% ± 2.3%) and 0.37 µM ConA (16.7% ± 4.8%), as shown in Figure 3A . When total numbers of cells expressing CD25 were compared, t