Efficacy of Carraguard-based microbicides in vivo despite variable in vitro activity

Abstract

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.

Figure 1. Carraguard can inhibit or enhance cell-free infection.

(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.

Figure 2. Carraguard augments mature DC-mediated amplification of infection.

(A) Mature moDCs were pre-incubated with 0, 10, or 200 µg/ml Carraguard and challenged with graded doses of Bal or RT-SHIV, washed, and co-cultured with TZM.bl cells. Mean SFCs (±SD, triplicate conditions) are shown from 1 of 4 experiments. (B) Carraguard-treated mature DCs were pulsed with Bal or RT-SHIV, washed, and co-cultured with TZM.bl cells as in (A). The percent inhibition of infection (mean±SD, triplicate conditions) is shown for 1 of 4 experiments. A statistically significant difference (p<0.05, two-tailed paired t-test) between the enhancement effects on Bal vs RT-SHIV infection is noted by the asterisk. (C) Mature DCs pre-treated with Carraguard (Pre, open squares) were challenged with 3000 TCID₅₀ of Bal or 4500 TCID₅₀ of RT-SHIV, washed, and co-cultured with TZM.bl cells. Alternatively, mature DCs were pulsed with virus, washed, added to TZM.bl cells and the graded doses of Carraguard added to the co-cultures (Post, filled squares). The percent inhibition (mean±SD, triplicates) are shown from 1 of 4 experiments. A statistically significant difference (p<0.01, two-tailed paired t-test) between the pre versus post Carraguard enhancement effects on Bal infection is noted by the asterisk.

Figure 3. Carraguard inhibits infection in immature DCs coincident with DC maturation.

(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).

Figure 4. MIV-150 overrides the augmentation of DC-driven infection by Carraguard.

(A) TZM.bl cells were pre-exposed to varying doses of MIV-150, challenged with the indicated viruses. Mean percent inhibition (±SD, triplicates) is shown from 1 of 3 experiments. (B) Mature DCs pre-treated with Carraguard (Carr Pre, open circles) or PC-817 (PC-817 Pre, open triangles; 33.3 nM MIV-150 per 2 µg/ml Carraguard) were pulsed with RT-SHIV, washed, and co-cultured with TZM.bl cells. Alternatively, mature DCs were pulsed with virus, washed, added to TZM.bl cells and the graded doses of Carraguard (Carr Post, filled circles) or PC-817 (PC-817 Post, filled triangles) added to the co-cultures. The concentrations indicated on the X axis indicate the Carraguard concentrations for each formulation. The percents of inhibition (mean±SD, triplicates) are shown for 1 of 3 similar experiments. (C) Mature DCs were treated with 2 µg/ml of Carraguard, pulsed with RT-SHIV, washed, and then cultured with TZM.bl cells in the presence (Carr+MIV-150) or absence (Carr) of varying doses of MIV-150 (nM). Percent inhibition of infection (mean±SD, triplicates) are shown for 1 of 3 identical experiments.

Figure 5. Carraguard-based gels inhibit vaginal infection in macaques.

Depo-Provera-treated animals were treated with 3 ml of MC, Carraguard (Carr), or PC-817 30 min prior to challenge with 1 ml of or 10³–10⁵ TCID₅₀ RT-SHIV. (A) Plasma viral loads were quantified by PCR and SIV gag RNA copies per ml of plasma are shown for each animal over time. The numbers of animals in the respective groups are indicated in each panel. Each symbol denotes a different animal (Table 1). (B) The frequencies (percentage of infected animals, mean±SEM) of infection in the MC, Carraguard, and PC-817-treated groups challenged with 10³ and 10⁴ TCID₅₀ are plotted. Carraguard (p<0.02) and PC-817 (p<0.03) significantly reduced the frequency of immunodeficiency virus infection compared to the MC-treated placebo group.

Figure 6. In vivo activity of MIV-150-containing MC gels.

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 Table 1.