Comparison of Tenofovir-Mediated Inhibition of HIV-1 and SIV Replication In Vitro
Having shown that tenofovir had no evidence of toxicity on TZM-bl cells, as assessed by MTT viability assay at the highest dose tested (unpublished data), we initially compared the activity of tenofovir, formulated as a solution from PMPA powder or formulated as a gel, as supplied by Gilead Sciences, against a panel of HIV-1 isolates with alternative secondary receptor usage (CXCR4-using RF, IIIB, and NL4.3; CCR5-using BaL, YU.2, and R8BaL) and against SIVmac251/32H; the latter being used for the macaque challenge experiments. There was no evidence of any difference in the mean average level of inhibition of SIVmac251/32H replication with either formulation at any dose (p = 0.89). This result was determined allowing for the inherent correlation across doses within the same intervention group using cross-sectional time series analysis. Furthermore, tenofovir IC50 values for SIVmac251/32H were of the same order of magnitude as those measured against both R5 and X4 restricted isolates of HIV ().
Solution and Gel Formulated Tenofovir Inhibited SIVmac251/32H Infectivity In Vitro at Similar Doses and in the Same Range as for Representative HIV-1 Isolates
Dosing and Outcome of Virus Challenge
We used a 1% tenofovir gel formulation made and supplied by Gilead Sciences as part of a programme on the development of a vaginal microbicide. Although this formulation had not been optimised for rectal use, we considered that it would provide a suitable starting point for preclinical evaluation. No adverse effects were seen following rectal gel administration. Virological analysis over a 20-wk period after rectal challenge with 20 MID50
showed that four of six animals in group A (tenofovir 15 min before challenge) and two of three animals in group D (tenofovir 2 h before challenge) were protected from systemic infection on the basis of failure to (a) recover virus from PBMC, (b) to detect proviral DNA in PBMC, (c) to detect vRNA in plasma, and (d) to detect SIV-specific antibodies in serum. All the naïve/not-dosed animals and three of four animals receiving placebo gel were infected and had high levels of circulating vRNA, and proviral DNA and virus was recoverable from the PBMC at all times of testing (). This protective effect just reached statistical significance at p
= 0.05 (). Furthermore, in comparison with the controls, two of three animals given tenofovir rectally prechallenge that became infected had modified virological outcomes. Virus was recovered from the PBMCs of animal D43 only at weeks two and six corresponding to the only times that vRNA was detected in plasma. Interestingly, the proviral DNA load remained below 50 copies/105
PBMC and by week 20, the last point of sampling, had declined to just above the limit of detection. In animal D56 the earliest time of virus detection by any of the methods used was 12 wk after challenge; a delay not seen in other animals in this study or in control animals rectally infected with the same virus stock in previous studies. Taken together, these data showed that tenofovir gel administered at least up to 2 h prior to virus challenge had a significant protective effect (p
= 0.003) (). Inclusion of another 17 naïve controls that had been challenged intrarectally with the same virus challenge stock [17
] revealed a highly significant level of protection from detectable virus infection (p
< 0.001) (). One of three animals given similar intrarectal dosing 2 h after virus challenge (group E) was protected from infection. Back titration of the virus challenge stock in C8166 cells confirmed infectivity titre had been maintained upon storage.
Rectal Administration of Tenofovir Gel Protected a High Proportion of Macaques against Subsequent Acquisition of SIV by Rectal Transmission
Contingency Table for Dosing and Outcome of Virus Challenge: Analysis for “Complete” Protection (i.e., No Virus Detection) Associated with Tenofovir Gel Administered Intrarectally Prior to Virus Challenge; p = 0.05
Table 3 Contingency Table for Dosing and Outcome of Virus Challenge: Analysis for Modified Outcome (i.e., “Complete” Protection or Reduced Frequency of Virus Detection) Associated with Tenofovir Gel Administered Intrarectally Prior to Virus Challenge; (more ...)
Table 4 Contingency Table for Dosing and Outcome of Virus Challenge: Analysis for “Complete” Protection, Including Historical Controls Challenged with the Same Virus Stock Intrarectally, Associated with Tenofovir Gel Administered Intrarectally (more ...)
Of the eight animals that remained VI/PCR negative, seven were clinically normal throughout the study, and one, D68, which had received placebo gel, whilst clinically normal at necropsy had enlarged axillary and inguinal nodes at week 12. In contrast, all of the animals in which virus was recovered at high frequency had some clinical signs and/or necropsy findings consistent with SIV infection, such as enlarged iliac, axillary and inguinal lymph nodes, and splenomegaly. One animal, D83, had evidence of progression to AIDS including loss of weight, tenting of skin, lung abnormalities including, grey discolouration, numerous petechial haemorrhages on the surface, and adhesions to the thoracic wall. Animal D43, which had been given tenofovir 15 min prior to virus challenge and showed only a weak and transient viraemia, remained clinically normal throughout the study.
To determine if virus was sequestered in tissue associated with the virus challenge, quantitative proviral PCR was used to examine MNC isolated from rectum and ileum, as well as iliac, inguinal, and mesenteric lymph nodes. In the apparently protected animals, there was no evidence of infection whereas in animal E81, a naïve challenged control, proviral loads of 26, 5, 130, 2,100, and 105 proviral DNA copies/105 MNC equivalents were detected in each tissue respectively.
T Cell Responses in Protected Animals
To determine if mucosal exposure to virus in the absence of overt infection had stimulated T cell immunity, SIV-specific IFN-γ ELISpot analysis was performed on PBMC from protected animals and one infected animal (E81) taken 20 wk after challenge. Four of seven protected animals had IFN-γ-secreting Gag-specific T cells at frequencies ranging from 144 to 261 SFU/106 cells, whereas the infected animal E81 had both Gag and Tat-specific circulating T cells (A). In contrast, no significant reactivity was seen in 12 naïve macaques tested (<40 SFU/106 cells). The finding of T cell responses in the virus exposed macaques was found to be statistically significant; p = 0.009 (Fisher exact test). Interestingly, the presence of Gag-specific IFN-γ secreting T cells in animal D68 (pre-exposed to placebo gel) confirmed that challenge virus gained access to antigen presenting cells. Interpretation of T cell reactivity in MNC from colorectal tissue was precluded by a very high spontaneous background; however, MNC from the small intestine of three of four protected animals contained SIV-specific IFN-γ-secreting T cells with broader antigen specificity (including specificity for nonstructural virus-encoded antigens) and in two of these cases no responses in PBMC (B). Thus the local mucosal immune system may have been primed by SIV antigens produced de novo during drug-modulated abortive or limited infection. Despite evidence of T cell priming, none of the protected animals had detectable SIV-specific serum antibodies (C).
SIV-Specific IFN-γ Secreting T Cells Were Detected in SIV-Challenged Macaques in the Absence of Serum Antibody Responses and Evidence of Overt Infection
Plasma Tenofovir Concentration and Protection Status
Analysis of plasma tenofovir concentration at the time of virus challenge, 15 min after gel administration, revealed a strong association with protective efficacy. The lowest concentration of plasma tenofovir associated with protection as defined by failure to isolate and/or detect virus in PBMC and plasma and lack of seroconversion, was 119.9 ng/ml (). Taking into account estimated plasma volume, protection was associated with as little as 0.11% of the total tenofovir applied. Moreover, an effect upon plasma viraemia was observed with as little as 0.06% of applied tenofovir detected in plasma at 15 min. In animals given tenofovir 2 h prior virus challenge plasma tenofovir, at the time of challenge ranged between below the 10 ng/ml limit of detection to 23.3 ng/ml. These results suggested therefore that drug concentration peaked rapidly after rectal dosing.
Plasma Tenofovir Levels and Association with Protection from Infection
Ex Vivo Modelling of Tenofovir Activity
To further address the possible mechanism of the protection observed we utilised our recently described in vitro colorectal explant model [31
] and adapted it to macaque tissue. Using tissues from another group of SIV-naïve macaques (group F), first, we demonstrated that SIVmac251/32H
replicated in this system and that replication was sensitive to addition of tenofovir in vitro (A). We also found similar replication in ileum–jejunum explants (unpublished data). Interestingly, replication in macaque explants peaked earlier (day seven) and at a lower level than that seen for HIV in human explants as determined by Gag p27/24 production (C. Herrera, unpublished data and [31
]), regardless of virus dose. Next, we investigated the replication kinetics in intestinal tissue explants taken from another four SIV naïve macaques that were given tenofovir gel in vivo 3 h prior to necropsy (group G). In colorectal explants from three of four animals, complete or nearly complete inhibition of virus replication was seen and in the other animal a high level of variability between replicate samples resulted in lower mean inhibition (B). In contrast, inhibition of virus replication was not seen in explants from the small intestine (unpublished data) suggesting that tenofovir was, at least in part, acting on cells at the virus portal of entry. Analysis of intestinal tissue samples collected at necropsy showed that all tenofovir-dosed animals had measurable concentrations of drug in lysates of colorectal tissue at concentrations between 20.8 and 54.2 μg/g protein but no drug was detected in lysates of homogenates from the small intestine (). Tissues from untreated animals (group F) acted as negative controls. To indirectly estimate the amount of intracellular phosphorylated tenofovir in tissues, samples were analysed with (to measure the combination of tenofovir + tenofovir monophosphate + tenofovir diphosphate) and without (to measure tenofovir only) phosphatase hydrolysis. Subtracting the concentration of tenofovir obtained from tissue samples without phosphatase, from the concentration of tenofovir obtained from tissue samples with phosphatase, demonstrated that between 46%–75% of total tenofovir in tissues was present as the intracellular mono- and diphosphate forms. On the basis of intracellular data describing tenofovir monophosphate:diphosphate ratios [32
], it was estimated that approximately 30%–60% of total tenofovir in tissues was present as the intracellular diphosphate form.
Colorectal Explants from Macaques Supported Replication of SIV That Was Inhibited by Pretreatment with Tenofovir In Vitro and In Vivo
Intracellular Concentrations of Base and Phosphorylated Tenofovir in Colorectal and Ileum–Jejunum of Macaques Given Tenofovir Gel Rectally