Epigallocatechin-3-gallate local pre-exposure application prevents SHIV rectal infection of macaques AbstractEpigallocatechin-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 SHIVSF162P3N. This protection has a per-exposure risk reduction of 91.5% (P鈥?鈥?.0009; log-rank test) and a complete protection of 87.5% (P鈥?lt;鈥?.001; Fisher鈥檚 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鈥? 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. IntroductionIn the absence of a protective vaccine or a cure for HIV-1 infection, intervention with highly potent and cost-effective inhibitors that target at the initial sites of HIV-1 infection is an ideal strategy for slowing the AIDS pandemic. Currently, HIV-1 infection spreads predominantly through sexual transmission, in which the virus gains access to a new host through the penile, rectal or vaginal mucosal tissues. During sexual intercourse, HIV-1-infected men transmit the virus through their semen, which carries free-floating virus, as well as the infected cells.1 Semen contains amyloid fibrils that potently enhance HIV-1 or SIV/SHIV infectivity in vitro.2,3,4 Therefore, to inhibit HIV-1 during sexual intercourse represents the most effective way in preventing transmission of the virus.Microbicides targeting the early events in sexual transmission of?thyc=12? HIV-1 have been extensively studied.5,6,7,8 Several recent studies reported that pre-exposure prophylaxis with the antiretrovirals (ARVs) was effective in the prevention of SHIV transmission in non-human primate (NHP) model.9,10 These ARVs-containing microbicides were usually used in the formulation of topical gels.7 However, the topical gel microbicides are associated with rectal mucosal damage, manifested as histological abnormality, and induction of proinflammatory response.11 In addition, ARVs-based microbicides are not effective on resistant HIV-1 strains and can induce viral resistance. Furthermore, semen can impair the antiviral efficacy of ARVs that act on intracellular targets at different steps of viral replication cycle.4 As compared with ARVs or other chemical drugs, the use of natural products for prevention of HIV-1 mucosal transmission might have some advantages, as they have low toxicity, minor side-effects, and are cost-effective.The consumption of green tea has many physiological and pharmacological health benefits. Mounting evidence has shown that epigallocatechin-3-gallate (EGCG), a natural and major ingredient of green tea, not only has anti-inflammatory and anti-oxidative effects, but also possesses antiviral abilities against diverse families of viruses, including HIV-1.12 In vitro studies have demonstrated that EGCG at a physiologic concentration could inhibit HIV-1 infection/replication in peripheral blood mononuclear cells (PBMCs) and macrophages.13 In addition, EGCG could remodel seminal amyloid fibrils14 and effectively suppress semen-mediated enhancement of HIV-1 replication.15 EGCG can interfere with several aspects of the HIV-1 life cycle, including the destruction of virions,16,17 the inhibition of reverse transcriptase and integrase activities of the virus.18,19 Most conclusively, several independent studies have confirmed that EGCG can bind to the same molecular pocket on CD4 receptor as does HIV-1 gp120, thus preventing the initial attachment of the viruses to CD4+ T cells.20,21 We recently reported that EGCG could also inhibit macaque SEVI (semen-derived enhancer of virus infection)-mediated enhancement of SIV/SHIV infection and degrade the formation of macaque semen-derived amyloid fibrils.22 A most recent clinical study showed that EGCG is a well-tolerated and non-toxic therapeutic agent that can upregulate protective CC chemokines against HIV-1.23 Taken together, these observations strongly argue for further studies, particularly in vivo investigations, on the anti-HIV-1 effect of EGCG as an entry inhibitor. In the present study, we evaluated whether mucosal pre-exposure application of EGCG can prevent SHIV rectal infection of rhesus macaques.ResultsEGCG inhibits HIV/SIV/SHIV in vitroThe major active ingredients of green tea are polyphenolic compounds, known as catechins, which include EGCG, EGC, ECG, and EC. EGCG accounts for 50% of the total green tea catechins. We first examined whether EGCG and other green tea catechins have the ability to inhibit infections with different strains of HIV-1, SHIV and SIV in TZM-bl cells. Among the green tea catechins, EGCG was the most potent inhibitor of the viruses (Fig.聽1a). In addition, EGCG dose-dependently inhibited HIV-1Bal replication in primary human macrophages (Fig.聽1b). Furthermore, EGCG could inhibit SHIV infection of primary lymphocytes and macrophages of macaques (Fig.聽1c). Evaluation of cytotoxicity revealed that the treatment of TZM-bl and PBMCs of macaques with the catechins for as long as 48鈥塰 at the dose as high as 100鈥壩糓 had little toxic effect (Supplementary Figures聽1 and 2).Fig. 1EGCG 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鈥塵in 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鈥塵in prior to HIV-1Bal 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鈥壩糓) for 10鈥塵in prior to SHIVSF162P3N infection. Intracellular gag RNA was measured by the real time PCR at day 5 post-infection. Data are shown as mean鈥壜扁€塖D, representative of three independent experiments with 3鈥? replicates. *P鈥?lt;鈥?.05, **P鈥?lt;鈥?.01 and ***P鈥?lt;鈥?.001Full size imageEGCG has little toxicity on rectal mucosaTo determine the in vivo protective effect of EGCG on SHIV rectal infection of macaques, we first assessed the toxicity of EGCG on rectal mucosa of macaques and mice. We demonstrated that the rectal application of 5鈥塵M EGCG for 10鈥塵in had little toxic effect on the rectal mucosa of macaques, as shown by the normal microstructures of epithelium, lamina propria, and intestinal glands in the rectum mucosa and submucosa (Supplementary Figure聽3a) and lack of fecal blood (Supplementary Table聽1). We also showed that EGCG had little effect on the expression of inflammatory cytokines and toxicity on the rectal mucosa of mice which is evidenced by normal intestinal microstructures, stool consistency, and lack of fecal blood (Supplementary Table聽2 and Figure聽3).EGCG protects macaques from rectal SHIV infectionWe next examined the protective effect of EGCG on repetitive and intra-rectal challenges with low-dose SHIVSF162P3N in macaques (Fig.聽2a). The assessment of the plasma SHIV RNA over the course of the study showed that all animals (8 out of 8) in the control group were infected, which required a median of 2.5 times of challenges (range from 1 to 8 times, Fig.聽2b). In contrast, only 1 out of 8 animals in the EGCG group became infected (Fig.聽2c). Evaluation of SHIV RNA and proviral DNA in PBMCs and the biopsied specimens (rectum and inguinal lymph nodes, LNs) at week 20 postinfection revealed infection in control animals, while no evidence of infection in 7 out of 8 EGCG-treated animals (Supplementary Figure聽4).Fig. 2EGCG protects macaques from intra-rectal SHIV infection. a Experimental design of EGCG protective effect on macaques. Sixteen male macaques were administered with 2鈥塵l of 5鈥塵M EGCG (8 animals) or 2鈥塵l of PBS (8 animals) atraumatically in rectum 10鈥塵in prior to each SHIVSF162P3N challenge. All animals were rectally inoculated with SHIVSF162P3N (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鈭?) 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 resultsFull size imageBecause gut-associated lymphoid tissue (GALT) is an important target organ of HIV-1 and may serve as a key viral reservoir, we also examined SHIV proviral DNA and RNA in the gastrointestinal tract (GI) tissues, lymph nodes (LNs) and intraepithelial lymphocytes (IELs) of study animals necropsied at week 36 postinfection. As shown in Fig.聽3 and Supplementary Table聽3, SHIV RNA and proviral DNA were detected in all analyzed tissues (GI tissues, LNs and IELs) from control animals. In contrast, only one animal (WRE08) in EGCG group had detectable SHIV RNA and DNA in the GI tissues, LNs and IELs. In addition to the plasma viral loads, we also examined plasma antibody against SHIV in the study animals. Consistent with the viral load data, while all control animals were positive for SHIV-specific antibody, only one animal (WRE08) in EGCG group had detectable SHIV antibody (Supplementary Table聽3). This animal had the highest CD69 and on CD4+ and CD8+ T cells in the first 2 weeks post-infection (Supplementary Figure聽5). In addition, there was a significant decrease of CD4+ T cell numbers in blood of this animal at week 3 after SHIV infection (Supplementary Figure聽6).Fig. 3SHIV 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鈥?鈥?鈥?) and EGCG group (green symbols, n鈥?鈥?) at necropsy. Log10 SHIV copies 2鈥壩糶鈭? 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 lymphocytesFull size imageStatistically, the EGCG treatment afforded a protective efficacy of 91.5% [1鈭?hazard ratio), P鈥?鈥?.0009, log-rank test] reduction in the per-exposure acquisition risk as compared with control (Fig.聽4). The complete protection for macaques in the EGCG group was 87.5% (P鈥?lt;鈥?.001, Fisher鈥檚 exact test).Fig. 4Protective efficacy of EGCG local pre-exposure application on SHIV rectal transmission in macaques. a Kaplan鈥揗eier plot demonstrating the protection in EGCG-treated animals (n鈥?鈥?) relative to PBS-treated animals (n鈥?鈥?). 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 test and Fisher鈥檚 exact testFull size imageMechanisms of EGCG-mediated in vitro and in vivo anti-HIV activitiesWe first studied the mechanisms of the in vitro findings that the pre-treatment of the target cells (TZM-bl, primary macaque lymphocytes and macrophages) with EGCG potently inhibited HIV/SHIV/SIV infection. As shown in Fig.聽5a, b, EGCG pretreatment of T cells could block the binding of CD4 antibody to CD4 receptor. We also observed that the incubation of CD4+ T cells with EGCG blocked the binding of FITC-conjugated HIV-1 gp120 to the cells (Fig.聽5c). To determine in vivo mechanism of EGCG-mediated protection of macaques from SHIV rectal infection, we examined the effect of EGCG on the infiltration and activation of T cells and macrophages in rectal mucosa (Fig.聽6a). EGCG rectal administration significantly reduced the infiltration of CD68+ macrophages (P鈥?lt;鈥?.01), but not CD3+ T cells in rectal mucosa (Fig.聽6b, c). In addition, EGCG pretreatment suppressed the local immune activation as shown by the diminished expression of CD163+ cells (P鈥?lt;鈥?.05) and HLA-DR+ cells (P鈥?lt;鈥?.01) in rectal mucosa (Fig.聽6d, e).Fig. 5Effect 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鈥塵in. 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鈥壜礛 of EGCG. The cells were then washed with PBS and further incubated with recombinant HIV-1 gp120 protein conjugated with FITC for 2鈥塰. The binding of gp120 to cells was indicated by intensity of FITC, which was examined by flow cytometry. Data are representative of three independent experimentsFull size imageFig. 6EGCG 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 脳200. b鈥?b>e Immunohistochemistry staining of the cell infiltration and chronic immune activation in rhesus macaques intra-rectally treated with PBS or EGCG. EGCG (5鈥塵M, 2鈥塵l) or PBS (2鈥塵l) was delivered to the rectum of rhesus macaques for 10鈥塵in prior to the challenge with SHIVSF162P3N (10 TCID50). Rectal tissues from two macaques were collected at autopsy (96鈥塰 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 in聽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 脳200. Data are shown as mean鈥壜扁€塖D, which were analyzed using the two-tailed Student鈥檚 t-test. *P鈥?lt;鈥?.05 and **P鈥?lt;鈥?.01Full size imageDiscussionWorldwide, sexual intercourse is currently the predominant mode of HIV-1 transmission, in which unprotected anal sex is riskier than unprotected vaginal sex.24 The rectum is a particularly susceptible entry site for HIV-1 or SHIV infection, as it is composed of only a single layer of columnar epithelium. In addition, the rectal mucosa contains numerous CD4-bearing target cells (CD4+ T cells, macrophages and DCs).25 The risk of HIV transmission during anal intercourse is estimated to be around 18 times greater than that in vaginal intercourse, according to the results of a meta-analysis.26 Therefore, the interventions to target the rectal site are critical in preventing sexual transmission of HIV-1. In this study, we demonstrate for the first time that EGCG can protect 7 out of 8 macaques from repeated intra-rectal challenges with SHIV, resulting in a per-exposure risk reduction of 91.5% (P鈥?i>=鈥?.0009 by the log-rank test) and a complete protection of 87.5% (P鈥?lt;鈥?.001 by Fisher鈥檚 exact test). All protected animals showed no evidence of systemic and mucosal SHIV infection as evidenced by the absence of viral RNA, DNA and antibodies. Interestingly, EGCG failed to protect one animal from SHIV infection. This animal appeared to be highly susceptible to SHIV challenge, as it became infected after only one-time of exposure to the virus. In contrast, all control animals except one required 2 or more repeated SHIV challenges (Fig.聽2b, c). In examining the immune activation markers (CD69 and HLA-DR) on CD4+ and CD8+ T cells of all infected animals, we found that this unprotected animal had the highest CD69 expression on CD4+ and CD8+ T cells among all infected monkeys in the first 2 weeks post-infection (Supplementary Figure聽5).EGCG has been reported to have in vitro antiviral effect on different subtypes, strains, and clinical isolates of HIV-1 in primary human CD4+ T cells and macrophages.13 We showed that EGCG had broad inhibitory effect on different strains of HIV-1, SIV, and SHIV in TZM-bl cells, macaque lymphocytes and macrophages (Fig.聽1). Studies from different laboratories have demonstrated that EGCG exhibits antiviral activity at multiple steps of HIV-1 infection/replication cycle, including the inhibition of HIV-1 reverse transcriptase,18 protease,16 and proviral genome integration.19 Importantly, EGCG-mediated HIV-1 inhibition appears to be its interference with HIV-1 entry into the target cells. EGCG can directly disassemble HIV-1 virion,16,17 block HIV-1 gp120 binding to CD4 molecule on T cells,20,21,27聽and degrade the seminal amyloid fibrils (a semen-derived enhancer of virus infection, SEVI) that reportedly enhances HIV-1 infection/transmission.2,4,14,28聽We found that EGCG specifically reduced the surface expression of CD4 receptor, but not CD3 receptor on human T cells (Fig.聽5a), suggesting that EGCG blocks the binding of CD4 antibody to CD4 receptor (Fig.聽5b). Also we showed that EGCG could block the binding of FITC-conjugated HIV-1 gp120 to CD4 receptor (Fig.聽5c). More importantly, we demonstrated that in vivo application of EGCG could suppress the macrophage infiltration/activation in the rectal mucosa of macaques. The protective effect of EGCG was also observed in a recent human study, showing that EGCG oral administration upregulated the CC chemokines (MIP-1伪/尾 and RANTES) against HIV-1.23 These combined in vitro and in vivo observations provide a possible mechanism for EGCG-mediated rectal protection from SHIV infection.These combined anti-HIV-1 actions of EGCG should reduce the risk of developing emergence of resistant viruses during the course of treatment. Furthermore, EGCG may confer an additional advantage over ARVs as it can counteract semen-mediated enhancement of HIV-1 infection.22,28 We recently reported that EGCG counteracted macaque SEVI-mediated enhancement of SIV/SHIV infection of macaque PBMCs.22 This finding is clinically relevant and important, as it has been reported that the anti-HIV-1 activity of a variety of potential microbicides could be compromised by the presence of semen or SEVI amyloid fibrils.4 Therefore, in order to identify the best candidates for the prevention of HIV-1 sexual transmission, it is necessary to evaluate the anti-HIV-1 efficacy of the ARVs-formulated microbicides in the presence of semen.29,29,30,31EGCG is generally considered a natural product with minimal toxicity. Clinically, EGCG has been found to be safe and well tolerated by the study subjects and the reported adverse events were rated as mild events.32,33 EGCG administration is safe in healthy individuals, at oral dose of 800鈥塵g per day over a time period of 4 weeks, which is equivalent to about 8鈥?6 cups of green tea a day.34 UIIman et al.35 reported that purified EGCG at the doses up to 1600 mg per day were generally well-tolerated and safe to use clinically. Oral application of EGCG in mouthwash formulation at the dose of 35鈥?7鈥塵M for 2鈥塵in daily for 7 days had little systemic side effects.36 A recent phase I clinical trial showed that the drug formulation of EGCG, Polyphenon E, is a well-tolerated, non-toxic therapeutic agent with no significant adverse effects at up to 1600 mg per day for 14 days.23 Our evaluation of cytotoxicity in the cell cultures showed that EGCG at doses up to 50鈥壩糓 had little cytotoxicity to TZM-bl cells and the primary macaque PBMCs. It was reported that the viability of HIV-infected human lymphocytes and macrophages was not significantly inhibited by EGCG over a range of 1鈥?00鈥壩糓.13 As compared to the concentrations of EGCG used in the cell cultures, higher dose (5鈥塵M) of EGCG was administered in the animal experiments. The rationale for use of this EGCG dose was based on the consideration that the macaque rectum contains multiple factors (feces, mucus, a number of microbial flora and metabolites) that could dilute and interfere with EGCG, reducing its antiviral activity. Nevertheless, we showed that 5鈥塵M EGCG had little toxicity and inflammatory effects on rectal mucosa of macaques and mice, as evidenced by the normal rectal epithelium structure and lack of inflammatory cytokine induction after the EGCG application (Supplementary Figure聽3, Tables聽1 and 2). The dosage of the FDA approved green tea extract Polyphenon E ointment (Veregen) for topical treatment of genital warts is about 100鈥?00鈥塵M.39,37,38,39Because sexual intercourse is a primary route of HIV-1 transmission worldwide and vaginal mucosal surface is a major site of the heterosexual transmission, future studies to determine the protective effect of EGCG on SHIV vaginal infection are also necessary. By extrapolating, it is likely that the same benefits of EGCG shown in the SHIV rectal infection model might apply to the prevention of vaginal SHIV infection. This supposition, however, will need to be confirmed experimentally given the differences between the rectal and vaginal transmission of HIV-1.25 The fact that EGCG is very stable in acidic solution,40 a condition similar to vaginal environment (pH range of 3.8鈥?.5), supports the future studies on use of EGCG for prevention of intravaginal HIV-1 transmission. Importantly, it is necessary to develop EGCG formulations that have stable, effective and long-lasting action on inactivation of HIV-1 in vaginal lumen and rectum. For example, EGCG in a buffered gel or film should be able to maintain an acidic pH in the presence of semen.Altogether with the known low toxicity, anti-inflammation and anti-viral properties of EGCG, our results argue in favor for more in vivo studies to examine the persistence, metabolism and chemical stability of EGCG at the mucosal exposure sites. These investigations are crucial for further clinical evaluation and development of EGCG as a safe, cost-effective and naturopathic microbicide for preventing sexual transmission of HIV-1, particularly in resource-poor settings.MethodsAnimalsSixteen adult male rhesus macaques were used to examine the protective effect of EGCG on prevention of SHIV rectal infection. The animals were obtained from the Kunming Institute of Zoology of Chinese Academy of Science. The animals were individually housed at the Animal Biosafety Level-III Laboratory at the Center for Animal Experiment of Wuhan University with AAALAC International accreditation (001274). All the animal protocols were approved by the Institutional Animal Care and Use Committee (approval number: 2015024) of Wuhan University in accordance with the NIH 鈥淕uide for the Care and Use of Laboratory Animals鈥? All the details of animal welfare were carried out in accordance with the recommendations of the Weatherall report, 鈥淭he Use of Nonhuman Primates in Research鈥? All the animals in this study were experimentally naive at the beginning and negative for antibodies against the specific pathogens. All the procedures were performed under anesthesia with ketamine, and necessary efforts were taken to minimize suffering, improve housing conditions, and provide enrichment opportunities for the study animals.Cells and virusesTZM-bl cells with integrated firefly luciferase gene under the control of HIV-1 LTR were obtained from the NIH AIDS Research Program and grown in DMEM medium. Human and macaque PBMCs were prepared by Ficoll gradient centrifugation and cultured in RPMI medium containing 10% FBS, 100鈥塙鈥塵l鈭? penicillin, 0.1鈥塵g鈥塵l鈭? streptomycin, 2鈥塵M L-glutamine and 20鈥塙鈥塵l鈭? interleukin-2聽(IL-2). Human PBMCs were incubated with complete DMEM in gelatin flasks for 45鈥塵in at 37鈥壜癈 to collect monocytes. After detachment with 10鈥塵M EDTA, monocytes were washed with DMEM and resuspended in complete DMEM and plated in 48-well plates. Monocytes differentiated into macrophages during in vitro culture. Macaque monocytes from PBMCs were cultured in vitro for 7 days when the cells became macrophages as described.41 SIVmac251, SIVmac239, SHIVKU-1, HIV-1Bal, and HIV-1NL4-3 strains were obtained from the NIH AIDS Research Program. The R5 SHIVSF162P3N derived from the HIV-1SF162 primary isolate42,43 was a generous gift from Dr. Cecilia Cheng-Mayer (Aaron Diamond AIDS Research Center, New York, NY) and was propagated in phytohemagglutinin (PHA)-activated macaque PBMCs. The 50% tissue culture infectious doses (TCID50) of SHIVSF162P3N was titrated in macaque PBMCs and the other virus stocks were titrated in TZM-bl cells.43 Briefly, PHA-activated PBMCs (2.5鈥壝椻€?05per well) were infected with a serial dilution of the virus stock in quadruplicate, starting with a 1/5 dilution in RPMI medium containing IL-2. The p27 antigens in culture supernatant were measured by SIV p27 ELISA (XpressBio, Frederick, MD). The TCID50 of the virus stocks were determined by the method of Reed and Muench.44Rectal administration of EGCG and SHIV challengeEGCG (Sigma-Aldrich, St. Louis, MO) was prepared as a stock solution (20鈥塵M) in PBS. The protective efficacy of EGCG on the rectal SHIV infection was evaluated in a repeated exposure macaque model using the SHIVSF162P3N, an extensively used SHIV strain in NHP model of sexual transmission.45,46 The animals were intramuscularly anesthetized with ketamine hydrochloride (10鈥塵g鈥塳g鈭?) plus xylazine hydrochloride (1鈥塵g鈥塳g鈭?). Anesthetized animals were maintained a ventral recumbent position and 2鈥塵l of EGCG (5鈥塵M) or PBS was administered atraumatically into the rectal vault prior to the intrarectal inoculation with SHIVSF162P3N at a dose of 10 TCID50.43 Animals that were not infected after the first inoculation were treated with EGCG or PBS and inoculated with SHIV again once biweekly. All the experiments were performed under highly controlled conditions by the same personnel using the same virus stock, inoculum dose and methods. Animals were considered infected and the virus challenges were stopped following two positive plasma SHIV RNA results. The experiments were stopped when all control animals became infected.9Plasma SHIV RNA determinationPlasma SHIV RNA was analyzed by real-time RT-PCR for SIV/SHIV gag gene as previously described.9 Primer used were: forward 5鈥?TGGAGAACAAAGAAGGATGTCAAA-3鈥? reverse 5鈥? CACCAGATGACGCAGACAGTATTAT-3鈥? Probe sequence was 6FAM-TTGGCACTAATGGAGCTAAGACCGAAAGTATT-BHQ1 (Gag-Btaq.2). Real-time PCR condition was: 95鈥壜癈 for 10鈥塵in, 40 cycles of 95鈥壜癈 for 30鈥塻 and 60鈥壜癈 for 15鈥塻. Duplicate samples were analyzed and the limit of detection (LOD) was 200 SIV or SHIV RNA copies ml鈭? plasma.PCR amplification of proviral DNA and RNADNA and RNA was extracted from PBMCs, lymphocytes isolated from LNs and other GI tissues (Supplementary Table聽3) with the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen, Valencia, CA). Specimens were analyzed in duplicate with 200 ng of DNA to amplify SHIV proviral DNA with the same primers and probes as for the plasma viral load assay described above. At week 20 after first SHIV challenge, inguinal LN and rectal mucosal were biopsied and subjected to RNA and proviral DNA detection. Intraepithelial lymphocytes from rectum or colon tissues were enriched to increase the sensitivity of the PCR as reported.9,47gp120 binding assayCD4+ T cells were incubated with 50鈥壩糓 of EGCG in RPMI-1640 medium for 10鈥塵in at 37鈥壜癈. Large volume of cooled (4鈥壜癈) PBS was then added immediately after incubation to halt the interaction between cells and EGCG. Cells were washed twice with PBS and aliquoted for two sets of experiments. In the first set of experiment, the cells were stained directly with PB-conjugated mAb against CD4 (Sk-3 clone) at room temperature subdue light for 45鈥塵in. PB-conjugated mouse IgG of unrelated specificity was used as a negative control. The stained cells were then examined by flow cytometry for CD4 expression. In the second set of experiments, the EGCG-treated CD4+ T cells were incubated with recombinant HIV gp120 protein conjugated with FITC (20鈥壩糶鈥塵l鈭?) for 1鈥塰. The cells were washed with PBS and the fluorescence intensity of gp120-FITC bound to the surface of CD4+ T cells was measured by the FACSCanto flow cytometer (BD Biosciences). Data were analyzed using the FlowJo data analysis software package (TreeStar Inc., USA).SerologyPlasma SHIV-specific antibodies (IgG and IgM) of the study animals were measured by enzyme immunoassay (EIA) as instructed by the manufacturer (Genscreen鈩?ULTRA HIV Ag-Ab; Bio-Rad).ImmunohistochemistryAutopsied specimens from the rectal tissues were obtained 4 days after the rectal administration of PBS or EGCG and fixed in paraformaldehyde. The sections were incubated with mouse monoclonal antibodies against CD3 (F7.2.38) or CD68 (DK25; Dako; Carpinteria, CA), CD163 (EDHu-1; AbD Serotec; Raleigh, NC), HLA-DR (LN3; eBioscience; San Diego, CA) overnight at 4鈥壜癈. Normal mouse serum was used as the negative antibody control for the specificity of the immunohistochemistry. The whole slides were digitally scanned using BX53 microscopy (Olympus, Japan) with ProScan III controller (Prior, UK). The tissue positivity for individual staining was assessed by Aperio ImageScope software (Leica Biosystems Inc., Buffalo Grove, IL).EGCG toxicity analysisThe in vitro cytotoxicity of EGCG on the TZM-bl and PBMCs was determined by MTT assay or by flow cytometry using 7-amino actinomycin D (7-AAD). The in vivo toxicity of EGCG on the rectal mucosa was examined by assessing the mucosal pathology, inflammatory response, stool consistency and bleeding.48,49 Briefly, EGCG was applied to rectum of the mice and the macaques. Fresh stools were then collected at 0, 24, 48, and 72鈥塰 post EGCG administration for occult blood test. Stool consistency of mice were graded accordingly as previously described.48 Inflammatory cytokines in the rectal tissues of the mice were measured by real time RT-PCR 72鈥塰 post-EGCG intrarectal administration. The impact of EGCG on rectal mucosal histology was analyzed by hematoxylin/eosin (HE) staining based on Obermeier鈥檚 procedure.50 The rectum was divided into 3 equal segments with different proximity to the anus (proximal, middle, and distal), cross sectioned, and fixed in 10% formalin overnight. The tissues were then embedded in paraffin and serially sectioned at a thickness of 5鈥壩糾. Histological analysis was performed and evaluated in a blinded fashion. Each score represented the mean of nine sections.Statistical analysisThe in vitro data were expressed as mean鈥壜扁€塻tandard deviation (SD) of triplicate cultures and statistical significance was assessed by Student鈥檚 t test. To protect the macaques from SHIVSF162P3N infection was analyzed using Cox proportional hazard models based on the exact partial likelihood for discrete time. The number of challenges was used as a discrete time scale. The hazard ratios with 95% confidence intervals (CI) for the per-exposure relative reductions of acquisition risk were calculated for EGCG group as compared to the control group. Protection against acquisition of infection was also analyzed by log-rank (Mantel-Cox) tests. The statistical analyses of complete protection for the study animals against SHIV challenge were performed by Fisher鈥檚 exact test. GraphPad Prism software (La Jolla, CA) was used for all statistical analyses. Significance was defined as *P鈥?lt;鈥?.05, **P鈥?lt;鈥?.01, and ***P鈥?lt;鈥?.001. References1.Ceballos, A. et al. Spermatozoa capture HIV-1 through heparan sulfate and efficiently transmit the virus to dendritic cells. J. Exp. Med. 206, 2717鈥?733 (2009).CAS聽 Article聽Google Scholar聽 2.Munch, J. et al. Semen-derived amyloid fibrils drastically enhance HIV infection. Cell 131, 1059鈥?071 (2007).Article聽Google Scholar聽 3.Usmani, S. M. et al. Direct visualization of HIV-enhancing endogenous amyloid fibrils in human semen. Nat. Commun. 5, 3508 (2014).Article聽Google Scholar聽 4.Zirafi, O. et al. Semen enhances HIV infectivity and impairs the antiviral efficacy of microbicides. Sci. Transl. Med. 6, 262ra157 (2014).Article聽Google Scholar聽 5.Cranage, M. et al. Prevention of SIV rectal transmission and priming of T cell responses in macaques after local pre-exposure application of tenofovir gel. PLoS Med. 5, e157 (2008).Article聽Google Scholar聽 6.Lederman, M. M. et al. Prevention of vaginal SHIV transmission in rhesus macaques through inhibition of CCR5. Science 306, 485鈥?87 (2004).CAS聽 Article聽Google Scholar聽 7.Shattock, R. J. Rosenberg, Z. Microbicides: topical prevention against HIV. Cold Spring Harb. Perspect. Med. 2, a007385 (2012).Article聽Google Scholar聽 8.Veazey, R. S. et al. Protection of rhesus macaques from vaginal infection by vaginally delivered maraviroc, an inhibitor of HIV-1 entry via the CCR5 co-receptor. J. Infect. Dis. 202, 739鈥?44 (2010).CAS聽 Article聽Google Scholar聽 9.Andrews, C. D. et al. Long-acting integrase inhibitor protects macaques from intrarectal simian/human immunodeficiency virus. Science 343, 1151鈥?154 (2014).CAS聽 Article聽Google Scholar聽 10.Andrews, C. D. et al. A long-acting integrase inhibitor protects female macaques from repeated high-dose intravaginal SHIV challenge. Sci. Transl. Med. 7, 270ra4 (2015). Google Scholar聽 11.Patton, D. L., Sweeney, Y. T. Paul, K. J. A summary of preclinical topical microbicide rectal safety and efficacy evaluations in a pigtailed macaque model. Sex. Transm. Dis. 36, 350鈥?56 (2009).Article聽Google Scholar聽 12.Steinmann, J., Buer, J., Pietschmann, T. Steinmann, E. Anti-infective properties of epigallocatechin-3-gallate (EGCG), a component of green tea. Br. J. Pharmacol. 168, 1059鈥?073 (2013).CAS聽 Article聽Google Scholar聽 13.Nance, C. L., Siwak, E. B. Shearer, W. T. Preclinical development of the green tea catechin, epigallocatechin gallate, as an HIV-1 therapy. J. Allergy Clin. Immunol. 123, 459鈥?65 (2009).CAS聽 Article聽Google Scholar聽 14.Castellano, L. M., Hammond, R. M., Holmes, V. M., Weissman, D. Shorter, J. Epigallocatechin-3-gallate rapidly remodels PAP85-120, SEM1(45-107), and SEM2(49-107) seminal amyloid fibrils. Biol. Open 4, 1206鈥?212 (2015).CAS聽 Article聽Google Scholar聽 15.Hartjen, P. et al. Assessment of the range of the HIV-1 infectivity enhancing effect of individual human semen specimen and the range of inhibition by EGCG. AIDS Res. Ther. 9, 2 (2012).Article聽Google Scholar聽 16.Fassina, G. et al. Polyphenolic antioxidant (-)-epigallocatechin-3-gallate from green tea as a candidate anti-HIV agent. AIDS 16, 939鈥?41 (2002).CAS聽 Article聽Google Scholar聽 17.Yamaguchi, K., Honda, M., Ikigai, H., Hara, Y. Shimamura, T. Inhibitory effects of (-)-epigallocatechin gallate on the life cycle of human immunodeficiency virus type 1 (HIV-1). Antivir. Res. 53, 19鈥?4 (2002).CAS聽 Article聽Google Scholar聽 18.Li, S., Hattori, T. Kodama, E. N. Epigallocatechin gallate inhibits the HIV reverse transcription step. Antivir. Chem. Chemother. 21, 239鈥?43 (2011).CAS聽 Article聽Google Scholar聽 19.Jiang, F. et al. The evaluation of catechins that contain a galloyl moiety as potential HIV-1 integrase inhibitors. Clin. Immunol. 137, 347鈥?56 (2010).CAS聽 Article聽Google Scholar聽 20.Kawai, K. et al. Epigallocatechin gallate, the main component of tea polyphenol, binds to CD4 and interferes with gp120 binding. J. Allergy Clin. Immunol. 112, 951鈥?57 (2003).CAS聽 Article聽Google Scholar聽 21.Williamson, M. P., McCormick, T. G., Nance, C. L. Shearer, W. T. Epigallocatechin gallate, the main polyphenol in green tea, binds to the T-cell receptor, CD4: potential for HIV-1 therapy. J. Allergy Clin. Immunol. 118, 1369鈥?374 (2006).CAS聽 Article聽Google Scholar聽 22.Zhou, R. H. et al. Epigallocatechin gallate inhibits macaque SEVI-mediated enhancement of SIV or SHIV infection. J. Acquir. Immune Defic. Syndr. 75, 232鈥?40 (2017).CAS聽 Article聽Google Scholar聽 23.Nance, C. L. et al. Translational medicine in HIV-1 infection: preclinical and clinical development of the green tea catechin, Epigallocatechin Gallate, as therapy and immunological signatures. J. Allergy Clin. Immunol. 139, AB209 (2017).Article聽Google Scholar聽 24.Jenness, S. M. et al. Unprotected anal intercourse and sexually transmitted diseases in high-risk heterosexual women. Am. J. Public Health 101, 745鈥?50 (2011).Article聽Google Scholar聽 25.Keele, B. F. Estes, J. D. Barriers to mucosal transmission of immunodeficiency viruses. Blood 118, 839鈥?46 (2011).CAS聽 Article聽Google Scholar聽 26.Baggaley, R. F., White, R. G. Boily, M. C. HIV transmission risk through anal intercourse: systematic review, meta-analysis and implications for HIV prevention. Int. J. Epidemiol. 39, 1048鈥?063 (2010).Article聽Google Scholar聽 27.Hamza, A. Zhan, C. G. How can (-)-epigallocatechin gallate from green tea prevent HIV-1 infection? Mechanistic insights from computational modeling and the implication for rational design of anti-HIV-1 entry inhibitors. J. Phys. Chem. B 110, 2910鈥?917 (2006).CAS聽 Article聽Google Scholar聽 28.Hauber, I., Hohenberg, H., Holstermann, B., Hunstein, W. Hauber, J. The main green tea polyphenol epigallocatechin-3-gallate counteracts semen-mediated enhancement of HIV infection. Proc. Natl Acad. Sci. USA 106, 9033鈥?038 (2009).CAS聽 Article聽Google Scholar聽 29.Roan, N. R. et al. The cationic properties of SEVI underlie its ability to enhance human immunodeficiency virus infection. J. Virol. 83, 73鈥?0 (2009).CAS聽 Article聽Google Scholar聽 30.Roan, N. R. Munch, J. Improving preclinical models of HIV microbicide efficacy. Trends Microbiol. 23, 445鈥?47 (2015).CAS聽 Article聽Google Scholar聽 31.Neurath, A. R., Strick, N. Li, Y. Y. Role of seminal plasma in the anti-HIV-1 activity of candidate microbicides. BMC Infect. Dis. 6, 150 (2006).Article聽Google Scholar聽 32.Chow, H. H. et al. Pharmacokinetics and safety of green tea polyphenols after multiple-dose administration of epigallocatechin gallate and polyphenon E in healthy individuals. Clin. Cancer Res. 9, 3312鈥?319 (2003).CAS聽 PubMed聽Google Scholar聽 33.Lambert, J. D. Yang, C. S. Mechanisms of cancer prevention by tea constituents. J. Nutr. 133, 3262S鈥?267S (2003).CAS聽 Article聽Google Scholar聽 34.Nguyen, M. M. et al. Randomized, double-blind, placebo-controlled trial of polyphenon E in prostate cancer patients before prostatectomy: evaluation of potential chemopreventive activities. Cancer Prev. Res. 5, 290鈥?98 (2012).CAS聽 Article聽Google Scholar聽 35.Ullmann, U. et al. A single ascending dose study of epigallocatechin gallate in healthy volunteers. J. Int. Med. Res. 31, 88鈥?01 (2003).CAS聽 Article聽Google Scholar聽 36.Yoon, A. J. et al. Topical application of green tea polyphenol (鈭?-Epigallocatechin-3-gallate (EGCG) for prevention of recurrent oral neoplastic lesions. J. Orofac. Sci. 4, 43鈥?0 (2012).CAS聽 Article聽Google Scholar聽 37.Lyseng-Williamson, K. A. Hoy, S. M. Polyphenon E 10% ointment: a guide to its use in the treatment of external genital and perianal warts. Drugs Ther. Perspect. 28, 6鈥? (2012). Google Scholar聽 38.Stockfleth, E. et al. Topical Polyphenon E in the treatment of external genital and perianal warts: a randomized controlled trial. Br. J. Dermatol. 158, 1329鈥?338 (2008).CAS聽 Article聽Google Scholar聽 39.Stalmach, A., Troufflard, S., Serafini, M. Crozier, A. Absorption, metabolism and excretion of Choladi green tea flavan-3-ols by humans. Mol. Nutr. Food Res. 53, S44鈥揝53 (2009).Article聽Google Scholar聽 40.Zhou, Q. et al. Investigating the stability of EGCg in aqueous media. Curr. Sep. 20, 83鈥?6 (2003).CAS聽Google Scholar聽 41.Sang, M., Liu, J. B., Dai, M., Wu, J. G. Ho, W. Z. Toll-like receptor 3 signaling inhibits simian immunodeficiency virus replication in macrophages from rhesus macaques. Antivir. Res. 112, 103鈥?12 (2014).CAS聽 Article聽Google Scholar聽 42.Shakirzyanova, M. et al. Pathogenic consequences of vaginal infection with CCR5-tropic simian-human immunodeficiency virus SHIVSF162P3N. J. Virol. 86, 9432鈥?442 (2012).CAS聽 Article聽Google Scholar聽 43.Harouse, J. M., Gettie, A., Tan, R. C., Blanchard, J. Cheng-Mayer, C. Distinct pathogenic sequela in rhesus macaques infected with CCR5 or CXCR4 utilizing SHIVs. Science 284, 816鈥?19 (1999).CAS聽 Article聽Google Scholar聽 44.Reed, L. J. Muench, H. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 493鈥?97 (1938). Google Scholar聽 45.Ren, W. et al. Mucosal transmissibility, disease induction and coreceptor switching of R5 SHIVSF162P3N molecular clones in rhesus macaques. Retrovirology 10, 9 (2013).CAS聽 Article聽Google Scholar聽 46.Tsai, L., Tasovski, I., Leda, A. R., Chin, M. P. Cheng-Mayer, C. The number and genetic relatedness of transmitted/founder virus impact clinical outcome in vaginal R5 SHIVSF162P3N infection. Retrovirology 11, 22 (2014).Article聽Google Scholar聽 47.Mehandru, S. et al. Primary HIV-1 infection is associated with preferential depletion of CD4鈥?鈥塗 lymphocytes from effector sites in the gastrointestinal tract. J. Exp. Med. 200, 761鈥?70 (2004).CAS聽 Article聽Google Scholar聽 48.Kim, J. J., Shajib, M. S., Manocha, M. M. Khan, W. I. Investigating intestinal inflammation in DSS-induced model of IBD. J. Vis. Exp. e3678 (2012).49.Tabet, S. R. et al. Safety and toxicity of nonoxynol-9 gel as a rectal microbicide. Sex. Transm. Dis. 26, 564鈥?71 (1999).CAS聽 Article聽Google Scholar聽 50.Obermeier, F. et al. Interferon-gamma (IFN-gamma)- and tumour necrosis factor (TNF)-induced nitric oxide as toxic effector molecule in chronic dextran sulphate sodium (DSS)-induced colitis in mice. Clin. Exp. Immunol. 116, 238鈥?45 (1999).CAS聽 Article聽Google Scholar聽 Download referencesAcknowledgementsThis work was funded by the National Science and Technology Major Projects of Infectious Disease (2012ZX10004501-001-004), the Mega-Projects of Science Research for the 12th Five-Year Plan, China (2014ZX10001003005), the National Natural Sciences Foundation of China (81271334, 81201261, 81301428), the Fundamental Research Funds for the Central Universities (2042017kf0030 to J.B.L.) and in part by the National Institutes of Health Grants (DA041302 and DA022177 to W.Z.H.; DA040329 and MH109385 to J.L.L.). HIV/AIDS Research Award from the Robert Mapplethorpe Foundation (New York, NY) to J.L.L. is also acknowledged. We thank C. Cheng-Mayer at Aaron Diamond AIDS Research Center for providing SHIVSF162P3N.Author informationAuthor notesThese authors contributed equally: J.B. Liu, J.L. Li.AffiliationsAnimal Biosafety Level III Laboratory at Center for Animal Experiment, Wuhan University School of Basic Medical Sciences, Wuhan, Hubei, 430071, ChinaJ. B. Liu,聽K. Zhuang,聽H. Liu,聽Q. H. Xiao,聽X. D. Li,聽R. H. Zhou,聽L. Zhou,聽T. C. Ma聽 聽W. Z. HoDepartment of Virology, Wuhan Centers for Disease Prevention and Control, Wuhan, Hubei, 430015, ChinaJ. L. Li,聽X. Wang,聽W. Zhou聽 聽M. Q. LiuDepartment of Pathology and Laboratory Medicine, Temple University Lewis Katz School of Medicine, Philadelphia, PA, 19140, USAW. Z. HoAuthorsJ. B. LiuView author publicationsYou can also search for this author in PubMed聽Google ScholarJ. L. LiView author publicationsYou can also search for this author in PubMed聽Google ScholarK. ZhuangView author publicationsYou can also search for this author in PubMed聽Google ScholarH. LiuView author publicationsYou can also search for this author in PubMed聽Google ScholarX. WangView author publicationsYou can also search for this author in PubMed聽Google ScholarQ. H. XiaoView author publicationsYou can also search for this author in PubMed聽Google ScholarX. D. LiView author publicationsYou can also search for this author in PubMed聽Google ScholarR. H. ZhouView author publicationsYou can also search for this author in PubMed聽Google ScholarL. ZhouView author publicationsYou can also search for this author in PubMed聽Google ScholarT. C. MaView author publicationsYou can also search for this author in PubMed聽Google ScholarW. ZhouView author publicationsYou can also search for this author in PubMed聽Google ScholarM. Q. LiuView author publicationsYou can also search for this author in PubMed聽Google ScholarW. Z. HoView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsJ.B.L., J.L.L., and W.Z.H. conceived of the study and experimental design and wrote the manuscript. J.B.L. and J.L.L. contributed equally to the work. J.B.L., J.L.L., H.L., Q.H.X., R.H.Z., and D.X.L. executed the macaque studies; X.W., K.Z., H.L., T.C.M., L.Z., and R.H.Z. performed viral loads and statistical analyses. M.Q.L. and W.Z. performed the SHIV antibody/plasma viral load measurement. J.B.L., J.L.L., and Q.H.X. performed IHC analyses and data analysis. All authors have read and approved the final version of this manuscript.Corresponding authorCorrespondence to W. Z. Ho.Ethics declarations Competing interests The authors declare no competing interests. Electronic supplementary material Supplementary InformationRights and permissionsReprints and PermissionsAbout this articleCite this articleLiu, J.B., Li, J.L., Zhuang, K. et al. Epigallocatechin-3-gallate local pre-exposure application prevents SHIV rectal infection of macaques. Mucosal Immunol 11, 1230鈥?238 (2018). https://doi.org/10.1038/s41385-018-0025-4Download citationReceived: 06 December 2017Revised: 23 February 2018Accepted: 27 March 2018Published: 31 May 2018Issue Date: July 2018DOI: https://doi.org/10.1038/s41385-018-0025-4