Efficacy of Carraguard®-Based Microbicides In Vivo Despite Variable In Vitro Activity

Anti-HIV microbicides are being investigated in clinical trials and understanding how promising strategies work, coincident with demonstrating efficacy in vivo, is central to advancing new generation microbicides. We evaluated Carraguard® and a new generation Carraguard-based formulation containing the non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 (PC-817). Since dendritic cells (DCs) are believed to be important in HIV transmission, the formulations were tested for the ability to limit DC-driven infection in vitro versus vaginal infection of macaques with RT-SHIV (SIVmac239 bearing HIV reverse transcriptase). Carraguard showed limited activity against cell-free and mature DC-driven RT-SHIV infections and, surprisingly, low doses of Carraguard enhanced infection. However, nanomolar amounts of MIV-150 overcame enhancement and blocked DC-transmitted infection. In contrast, Carraguard impeded infection of immature DCs coincident with DC maturation. Despite this variable activity in vitro, Carraguard and PC-817 prevented vaginal transmission of RT-SHIV when applied 30 min prior to challenge. PC-817 appeared no more effective than Carraguard in vivo, due to the limited activity of a single dose of MIV-150 and the dominant barrier effect of Carraguard. However, 3 doses of MIV-150 in placebo gel at and around challenge limited vaginal infection, demonstrating the potential activity of a topically applied NNRTI. These data demonstrate discordant observations when comparing in vitro and in vivo efficacy of Carraguard-based microbicides, highlighting the difficulties in testing putative anti-viral strategies in vitro to predict in vivo activity. This work also underscores the potential of Carraguard-based formulations for the delivery of anti-viral drugs to prevent vaginal HIV infection

A Macaque Model to Study Vaginal HSV-2/Immunodeficiency Virus Co-Infection and the Impact of HSV-2 on Microbicide Efficacy

Herpes simplex virus type-2 (HSV-2) infection enhances the transmission and acquisition of human immunodeficiency virus (HIV). This occurs in symptomatic and asymptomatic stages of HSV-2 infection, suggesting that obvious herpetic lesions are not required to increase HIV spread. An animal model to investigate the underlying causes of the synergistic action of the two viruses and where preventative strategies can be tested under such complex physiological conditions is currently unavailable.We set out to establish a rhesus macaque model in which HSV-2 infection increases the susceptibility to vaginal infection with a model immunodeficiency virus (simian-human immunodeficiency virus, SHIV-RT), and to more stringently test promising microbicides. HSV-2 exposure significantly increased the frequency of vaginal SHIV-RT infection (n = 6). Although cervical lesions were detected in only approximately 10% of the animals, long term HSV-2 DNA shedding was detected (in 50% of animals followed for 2 years). Vaginal HSV-2 exposure elicited local cytokine/chemokine (n = 12) and systemic low-level HSV-2-specific adaptive responses in all animals (n = 8), involving CD4(+) and CD8(+) HSV-specific T cells (n = 5). Local cytokine/chemokine responses were lower in co-infected animals, while simian immunodeficiency virus (SIV)-specific adaptive responses were comparable in naïve and HSV-2-infected animals (n = 6). Despite the increased frequency of SHIV-RT infection, a new generation microbicide gel, comprised of Carraguard(R) and a non-nucleoside reverse transcriptase inhibitor MIV-150 (PC-817), blocked vaginal SHIV-RT infection in HSV-2-exposed animals (n = 8), just as in naïve animals.We established a unique HSV-2 macaque model that will likely facilitate research to define how HSV-2 increases HIV transmission, and enable more rigorous evaluation of candidate anti-viral approaches in vivo

An Antiretroviral/Zinc Combination Gel Provides 24 Hours of Complete Protection against Vaginal SHIV Infection in Macaques

Repeated use, coitus-independent microbicide gels that do not contain antiretroviral agents also used as first line HIV therapy are urgently needed to curb HIV spread. Current formulations require high doses (millimolar range) of antiretroviral drugs and typically only provide short-term protection in macaques. We used the macaque model to test the efficacy of a novel combination microbicide gel containing zinc acetate and micromolar doses of the novel non-nucleoside reverse transcriptase inhibitor MIV-150 for up to 24 h after repeated gel application.Rhesus macaques were vaginally challenged with SHIV-RT up to 24 h after repeated administration of microbicide versus placebo gels. Infection status was determined by measuring virologic and immunologic parameters. Combination microbicide gels containing 14 mM zinc acetate dihydrate and 50 µM MIV-150 afforded full protection (21 of 21 animals) for up to 24 h after 2 weeks of daily application. Partial protection was achieved with the MIV-150 gel (56% of control at 8 h after last application, 11% at 24 h), while the zinc acetate gel afforded more pronounced protection (67% at 8-24 h). Marked protection persisted when the zinc acetate or MIV-150/zinc acetate gels were applied every other day for 4 weeks prior to challenge 24 h after the last gel was administered (11 of 14 protected). More MIV-150 was associated with cervical tissue 8 h after daily dosing of MIV-150/zinc acetate versus MIV-150, while comparable MIV-150 levels were associated with vaginal tissues and at 24 h.A combination MIV-150/zinc acetate gel and a zinc acetate gel provide significant protection against SHIV-RT infection for up to 24 h. This represents a novel advancement, identifying microbicides that do not contain anti-viral agents used to treat HIV infection and which can be used repeatedly and independently of coitus, and underscores the need for future clinical testing of their safety and ability to prevent HIV transmission in humans

Identification of personal lubricants that can cause rectal epithelial cell damage and enhance HIV type 1 replication in vitro

Over-the-counter personal lubricants are used frequently during vaginal and anal intercourse, but they have not been extensively tested for biological effects that might influence HIV transmission. We evaluated the in vitro toxicity anti-HIV-1 activity and osmolality of popular lubricants. A total of 41 lubricants were examined and compared to Gynol II and Carraguard as positive and negative controls for toxicity, respectively. Cytotoxicity was assessed using the XTT assay. The MAGI assay with R5 and X4 HIV-1 laboratory strains was used to evaluate antiviral activity. The effect of the lubricants on differentiated Caco-2 cell monolayers (transepithelial electrical resistance, TEER) was also measured. None of the lubricants tested showed significant activity against HIV-1. Surprisingly, four of them, Astroglide Liquid, Astroglide Warming Liquid, Astroglide Glycerin & Paraben-Free Liquid, and Astroglide Silken Secret, significantly enhanced HIV-1 replication (

The nonnucleoside reverse transcriptase inhibitor MIV-150 in carrageenan gel prevents rectal transmission of simian/human immunodeficiency virus infection in macaques

Development of a microbicide that prevents rectal transmission of human immunodeficiency virus (HIV) is a vital component in reducing HIV spread. We recently demonstrated that a formulation of the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 in carrageenan reduced vaginal infection of macaques with simian immunodeficiency virus SIVmac239 with HIV-1 HxB2 reverse transcriptase (SHIV-RT). Herein, we performed the first testing of MIV-150-carrageenan against rectal infection. Rhesus macaques were treated rectally with MIV-150-carrageenan or methyl cellulose (MC) placebo gel up to 4 h prior to rectal challenge with 10 3 or 10 4 50% tissue culture infective doses (TCID 50) of SHIV-RT. Infection was assessed by measuring plasma virus RNA as well as T and B cell responses. MIV-150-carrageenan protected all animals challenged with 10 3 TCID 50 when gel was applied either 30 min or 4 h prior to challenge, while 100% of the MC-treated animals became infected (n = 4 each; P \u3c 0.03). Partial protection (2 of 4 animals) by MIV-150-carrageenan was observed for rectal challenge with 10-fold more virus applied 4 h after the gel. Sequencing of the RT gene from plasma virus RNA isolated at peak viremia confirmed that both of these animals (like infected MC controls) were infected with wild-type virus. Infection correlated with the development of SIV-specific T and B cell responses. MIV-150 was detected in the rectal fluids and tissues 4 h after gel application but was not detected in the blood at any time (0.5 to 24 h). These data are promising for the development of NNRTIcontaining gels to prevent rectal HIV transmission

Carraguard inhibits infection in immature DCs coincident with DC maturation.

<p>(A) Immature DCs were pre-incubated with graded doses of Carraguard, after which the DCs were challenged with Bal (down triangles) or HIV Δenv pseudotyped with the VSVg envelope (Δenv, filled squares). Cells were harvested 5 d later and stained for (A) HIV capsid p24 protein (mAb KC-57-RD1) or (B) the surface maturation markers CD83 and CD86. (A) Percent inhibition (mean±SD, triplicates) of infection and (B) the MFIs (mean±SD of triplicates) of CD83 (black bar) and CD86 (grey bar) expression (on the entire DC population) are shown for 1 of 4 replicate experiments. CD83 and CD86 up-regulation in response to increasing doses of Carraguard correlate closely (r = 0.99). (C) p24 expression is plotted against CD83, showing the correlation between lower CD83 levels and HIV infection. Comparable results were obtained when comparing CD86 and p24 expression (data not shown).</p

<i>In vivo</i> activity of MIV-150-containing MC gels.

<p>Depo-Provera-treated animals were treated with (A) 3 ml of MC containing 500 µM MIV-150 30 min prior to vaginal challenge with 10³ or 10⁴ TCID₅₀ of RT-SHIV or (B) 3 ml of MC containing 500 µM MIV-150 24 h before, 30 min before, and 24 h after vaginal challenge with 10⁴ TCID₅₀ of RT-SHIV. Plasma viral loads over time are shown for the indicated numbers of animals in each group. One year after challenge the 3 animals with the low-level initial infection (now with undetectable virus) and two of the normally infected animals were treated with the anti-CD8 mAb to deplete CD8 cells. (C) Effective depletion of CD8 cells was verified by flow cytometry and the CD8 cells per µl of blood are shown for each animal. (D) Analysis of the plasma virus loads before during and after CD8 depletion, revealed no rebound in virus levels in the 3 animals with the unusual acute low-level infection. Each symbol denotes a different animal that are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003162#pone-0003162-t001" target="_blank">Table 1</a>.</p

Carraguard can inhibit or enhance cell-free infection.

<p>(A) TZM.bl cells were exposed to 0–10 µg/ml of Carraguard, before graded doses of HIV_Bal or RT-SHIV were added. 24 h later, the media was replaced and cells were cultured for 4 d. The numbers of β-gal expressing SFCs per well are shown (mean±SD, triplicate cultures). (B) Titrated amounts of Carraguard were tested against of HIV_MN (MN, up triangles), HIV_Bal (Bal) (down triangles), or RT-SHIV (circles) in the TZM.bl cell line as in (A). The data are shown as the percent inhibition (mean±SD, triplicate cultures) of infection in the test conditions relative to the no Carraguard control. Negative % inhibition values represent enhancement, with no inhibitor effect at 0%. (C) Titrated amounts of Carraguard were added to PHA activated PBMCs before the cells were cultured with Bal or RT-SHIV for 5 d. Infection was measured by Q-PCR. Data are shown for triplicate cultures (mean±SD). Data in (A–C) are representative of 3 independent experiments with different donors in each case.</p