Epigallocatechin-3-gallate local pre-exposure application prevents SHIV rectal infection of macaques

Abstract

Epigallocatechin-3-gallate (EGCG), a natural and major ingredient of green tea, has been shown to have anti-inflammation and anti-HIV-1 properties. We demonstrated that the intrarectal administration of EGCG could protect rhesus macaques from repetitive, intrarectal challenges with low-dose SHIV_SF162P3N. This protection has a per-exposure risk reduction of 91.5% (P = 0.0009; log-rank test) and a complete protection of 87.5% (P < 0.001; Fisher's exact test). All protected animals showed no evidence of systemic and mucosal SHIV infection as demonstrated by the absence of viral RNA, DNA and antibodies. In contrast, all controls became infected after repeated SHIV challenges (a median of 2.5 times, range of 1-8 times). Mechanistically, EGCG could block the binding of HIV-1 gp120 to CD4 receptor and suppress the macrophage infiltration/activation in the rectal mucosa of macaques. These data support further clinical evaluation and development of EGCG as a novel, safe and cost-effective microbicide for preventing sexual transmission of HIV-1.

Figure 1

EGCG inhibits viral infectivity of a broad spectrum of AIDS-related viruses. (a) TZM-bl cells were treated with the indicated concentrations of green tea-derived catechins (EC, EGC, ECG and EGCG) for 10 min prior to infection with different strains of HIV-1 (Bal, NL4-3), SHIV (SF162P3N, KU-1), or SIV (mac239, mac251). Viral infectivity was assessed by luciferase activity, which is expressed as a percentage relative to that of the control (untreated). The half maximal inhibitory concentration (IC₅₀) of EGCG is indicated, which was calculated based on the untreated control by the method of Reed and Muench. (b) Human peripheral blood monocyte-derived macrophages were incubated with the indicated doses of EGCG for 10 min prior to HIV-1_Bal infection. Culture supernatant was collected on day 7 post-infection for HIV-1 reverse transcriptase (RT) assay. Cellular RNA was subjected to the real time RT-PCR for HIV-1 gag and GAPDH RNA. Data are expressed as HIV-1 RNA levels relative (%) to untreated control, which is defined as 100%. (c) Primary lymphocytes and macrophages from rhesus macaques were treated with or without EGCG (50 μM) for 10 min prior to SHIVSF162P3N infection. Intracellular gag RNA was measured by the real time PCR at day 5 post infection. Data are shown as mean ± SD, representative of three independent experiments with 3-4 replicates. *P<0.05, **P<0.01 and ***P<0.001.

Figure 2

EGCG protects macaques from intra-rectal SHIV infection. (a) Experimental design of EGCG protective effect on macaques. Sixteen male macaques were administrated with 2 ml of 5 mM EGCG (8 animals) or 2 ml of PBS (8 animals) atraumatically in rectum 10 min prior to each SHIV_SF162P3N challenge. All animals were rectally inoculated with SHIV_SF162P3N (10 TCID₅₀) for up to 8 times or until infection occurred. All animals were biopsied at week 20 and necropsied at week 36 postinfection for the evaluation of SHIV RNA and proviral DNA in the multiple tissues. (b, c) Longitudinal assessment of the plasma SHIV RNA (copies ml^-1) levels in the animals with intrarectal pretreatment with PBS (control) or EGCG prior to SHIV challenges (up to 8 times or till infection occurred). Duplicate plasma samples were analyzed for SHIV RNA detection. Animals were considered infected and the virus challenges were stopped following two consecutive positive plasma SHIV RNA results.

Figure 3

SHIV RNA and DNA detection in multiple tissues necropsied at week 36 post first SHIV challenge. SHIV RNA (a) and DNA (b) assays in the indicated tissues from the animals of PBS control (red symbols, n=6∼8) and EGCG group (green symbols, n=8) at necropsy. Log SHIV copies 2 μg^-1 total genomic RNA or DNA equivalents are shown. Symbols represent individual animals and are pooled from three independent experiments. Triplication tissue samples were conducted in each independent experiment. The dot line: the detection threshold. GI: gastrointestinal tract; LN: lymph nodes; Jej-Mes: jejunal mesenteric; Col-Mes: colonal mesenteric; IEL: intraepithelial lymphocytes.

Figure 4

Protective efficacy of EGCG local pre-exposure application on SHIV rectal transmission in macaques. (a) Kaplan-Meier plot demonstrating the protection in EGCG-treated animals (n=8) relative to PBS-treated animals (n=8). (b) Statistical analyses include the hazard ratio with 95% confidence interval and the per-exposure reduction of acquisition risk in each group, with P values reflecting log-rank tests and Fisher's exact test.

Figure 5

Effect of EGCG on binding of anti-CD4 antibody and gp120 to CD4 T cells. (a) Peripheral blood mononuclear cells were incubated with indicated doses of EGCG for 10 min. The reaction was then terminated with cold PBS. Cells were washed and then stained with anti-CD3 and anti-CD4 antibodies and subjected to flow cytometry analysis. The data shown are representative of three experiments with cells from three different donors. (b) Overlapping of the CD4 expression in PBMCs pretreated with indicated concentrations of EGCG. (c) Interference of binding of HIV-1 gp120 to CD4⁺ T cells. Purified CD4⁺ T cells were incubated with or without (w/o) 50 μM of EGCG. The cells were then washed with PBS and further incubated with recombinant HIV-1 gp120 protein conjugated with FITC for 2 h. The binding of gp120 to cells was indicated by intensity of FITC, which was examined by flow cytometry. Data are representative of three independent experiments.

Figure 6

EGCG suppresses macrophage infiltration and immune activation in the rectal mucosa of SHIV-infected macaques. (a) Morphological observation of the microstructure of the rhesus macaque rectum. Architecture of the rectal mucosa of rhesus macaques as examined by hematoxylin and eosin staining. Magnification ×200. (b-e) Immunohistochemistry staining of the cell infiltration and chronic immune activation in rhesus macaques intra-rectally treated with PBS or EGCG. EGCG (5 mM, 2 ml) or PBS (2 ml) was delivered to the rectum of rhesus macaques for 10 min prior to the challenge with SHIV_SF162P3N (10 TCID₅₀). Rectal tissues from two macaques were collected at autopsy (96 h post-infection). Cell infiltrates were demonstrated by immunohistochemistry staining with anti-CD3 (b) or anti-CD68 (c) antibody. Tissue activation was examined by staining with anti-CD163 (d) or anti-HLA-DR (e) antibody. The positivity of mucosal tissue for CD3⁺, CD68⁺, CD163⁺, and HLA-DR⁺ cells in the rectum mucosa were quantified using the Aperio Image Scope software. The solid yellow lines (d, e) were used to designate regions, including the epithelium, lamina propria and intestinal glands of the rectal mucosa and submucosa, for algorithm analysis. Dotted yellow lines excluded the enclosed regions for the calculation. The positive cells within the mucosa and submucosa were counted per high-power field. At least two cross-sectioned rectal segments with different proximity to the anus were scanned and analyzed. Original magnification ×200. Data are shown as mean ± SD, which were analyzed using the 2-tailed Student's t-test. *P<0.05 and **P <0.01.