We demonstrate for the first time that high mucosal and systemic antiretroviral drug concentrations in cells that are primary targets for HIV infection do not necessarily translate into high prophylactic efficacy. As expected from the high cellular permeation of GS7340, intracellular TFV-DP levels in PBMCs and lymphoid tissues were much higher with GS7340 than with TDF despite the lower GS7340 dose used (8
). TFV-DP concentrations in PBMCs remained high for as long as 7 days, with levels that exceeded by 100-fold those seen at peak with TDF. In rectal lymphocytes, TFV-DP levels at 3 days were similarly high. Despite the favorable drug PK profile and high tissue TFV-DP levels, GS7340 did not protect macaques from rectal SHIV exposure. We relate these findings to intracellular drug pharmacodynamics in the rectal mucosa by showing that rectal lymphocytes have a high dATP content that likely rendered TFV-DP less effective in blocking early infection events (1
). We also found that infection outcome was associated with dATP/TFV-DP ratios in PBMCs. Our results identify dATP/TFV-DP ratios as a negative correlate of protection with tenofovir and suggest that for nucleoside RT inhibitors, drug activity and prophylactic efficacy will depend on natural substrate concentrations at mucosal sites. Since high systemic TFV-DP levels that were sufficient to blunt acute viremias did not result in protection, our findings also suggest that active drug concentrations at the mucosa are more critical for PrEP effectiveness.
The finding of high dATP levels in rectal tissues was not completely surprising, since immune effector sites are in a state of physiological inflammation that results in cell activation and increased dNTP pools (1
). In PBMCs, phytohemagglutinin (PHA) stimulation increases intracellular dNTP concentrations 4- to 13-fold (7
). Since most of the CD4+
cells in the gut are CCR5+
activated memory CD4+
cells and are thus preferred cellular targets for HIV and SIV infection, high dATP pools might have reduced the ability of TFV-DP to block reverse transcription and prevent rectal infection (2
). In contrast, a lower dATP content in PBMCs and less availability of activated target cells might increase the effectiveness of TFV against parenteral exposures. These two possible scenarios were noted in macaques receiving prophylactic treatment with oral TDF or subcutaneous TFV. While daily oral TDF did not prevent rectal infection during repeated exposures to SHIVSF162P3
, subcutaneous TFV successfully protected macaques parenterally exposed to a high dose of SIVmne
). It will also be important to determine how high dATP levels are in vaginal lymphocytes and if they can be affected by inflammation associated with sexually transmitted diseases (16
). Additional studies comparing TFV-DP levels achieved in vaginal and rectal tissues after oral dosing, comparing dATP contents in rectal and vaginal lymphocytes, and assessing how all these relate to efficacy in preventing infection will provide valuable information on potential pharmacodynamic and chemoprophylactic differences between these two compartments.
In contrast to levels in circulating lymphocytes and lymphoid tissues, levels of TFV-DP in rectal tissues at 3 days were similar with GS7340 and TDF dosing. In both instances, high intracellular TFV-DP concentrations were associated with high extracellular TFV levels in rectal secretions. Such high TFV levels may originate partially from degradation of GS7340 or TDF to TFV by esterases present in the intestines and/or from trapping of TFV in mucus (22
). The lack of protection seen despite such high tissue TFV-DP concentrations is worrisome and suggests that the threshold for protection against rectal transmission by tenofovir alone may be very high. This observation may also explain why the combination of TDF and FTC was more protective than TDF alone in our animal model, and it suggests that the additional antiviral activity provided by FTC is essential for the efficacy of PrEP against rectal transmission (11
Several important observations can be made from the PrEP breakthrough infections. First, acute viremias were blunted in the 4 PrEP animals who had breakthrough infections despite receiving only one weekly dose. The low viremias seen in these macaques demonstrate clearly the potent systemic antiviral activity of GS7340 and can be explained by the persistently high intracellular TFV-DP concentrations. These virologic responses are consistent with the 1.8 log10
reduction in plasma virus loads seen in humans during GS7340 treatment (26a
). The rapid decline in virus loads to undetectable levels within 4 to 8 weeks might also explain the lack of selection of the K65R mutation associated with TFV resistance. Similar blunted viremias have been noted in PrEP breakthrough infections with FTC or Truvada and have also been associated with reduced risks of resistance emergence (8
). Interestingly, all infections were initiated with WT viruses, suggesting that initial virus replication originates from cells that are not protected by PrEP either because of suboptimal TFV-DP concentrations or, possibly, because of high intracellular dATP levels in gut lymphocytes.
Several important limitations to our study should be considered. First, we did not measure TFV-DP concentrations in cellular subpopulations from rectal tissues that are also primary targets during early infection and might be inadequately protected by GS7340, such as macrophages or dendritic cells (25
). Different molecular transport pathways for GS7340 and TFV might result in variable cellular drug exposures after GS7340 or TDF dosing. Second, our analysis of dATP contents in rectal lymphocytes was done 25 to 32 weeks after SHIV infection. It is not known whether the infection had contributed to the high dATP levels seen in rectal lymphocytes from these animals, although high dATP concentrations are consistent with the activation status of rectal lymphocytes (1
). Also, SHIVSF162P3
infections are not highly pathogenic; most animals appear to control infection (13
). Our analysis of dATP levels in one uninfected, untreated macaque suggests that neither infection status nor drug treatment might be responsible for the high dATP levels seen in our animals, although this observation needs to be further confirmed with more uninfected macaques. It will also be important to find out whether the high dATP contents seen in rectal lymphocytes from rhesus macaques are also observed in the gastrointestinal tracts of humans.
In summary, we show that GS7340 efficiently delivers TFV into peripheral lymphocytes, lymphoid tissues, and rectal tissue. However, we demonstrate that high rectal and systemic TFV exposure was not sufficient to prevent rectal SHIV transmission in macaques. We explain this paradox by showing high dATP concentrations in rectal mononuclear cells, which may reduce the ability of TFV-DP to block early infection events. Our results identify dATP/TFV-DP ratios as a negative correlate of protection by TFV and suggest that natural substrate concentrations in mucosal target cells may potentially modulate the prophylactic efficacy of tenofovir and may have broader implications for the entire nucleotide RT inhibitor drug class. Pharmacodynamic differences between rectal, vaginal, and systemic tissues may possibly impact the prophylactic efficacy of antiretroviral drugs against distinct routes of HIV transmission.