In this study, we demonstrate that an amyloid-binding small molecule is an efficient inhibitor of SEVI- and semen-mediated enhancement of HIV infectivity. We chose to examine the amyloid-binding small molecule BTA-EG6
based on the hypothesis that it would effectively bind and coat SEVI fibrils as it has been previously shown to do for Aβ (6
). We found that BTA-EG6
bound to the SEVI fibrils and interfered with their ability to enhance HIV infectivity, suggesting that other amyloid-binding small molecules might also prove to be effective microbicide candidates targeting SEVI. Importantly, BTA-EG6
did not have any direct inhibitory effects on the infectivity of HIV-1 alone.
BTA-EG6 inhibited SEVI-mediated enhancement of infection by both X4 (HIV-1IIIB) and R5 (HIV-1ADA) strains, in a dose-dependent fashion. In the case of HIV-1ADA, we calculated the IC50 to be 13 μm; this value is 100-fold higher than the measured Kd of BTA-EG6 for binding to aggregated SEVI peptides (127 nm). One explanation for this difference is that the ability of BTA-EG6 to compete with virion/fibril or virion/cell interactions requires a greater number of BTA-EG6 molecules than the noncompetitive binding of BTA-EG6 to SEVI alone.
also inhibited SEVI-enhanced infection of primary cells (human peripheral blood mononuclear cells) in a dose-dependent fashion, and it blocked SEVI-enhanced binding of X4 (HIV-1IIIB
) and R5 (HIV-1ADA
) strains to target cells (including both Jurkat T cells and A2En endocervical cells). The ability of SEVI to enhance virus binding to endocervical cells suggests an additional mechanism by which these fibrils might enhance HIV-1 transmission in vivo
. The fibrils may enhance the attachment of virus to cells at the cervicovaginal barrier, thereby increasing the likelihood of productive infection of viral target cells in the cervicovaginal mucosa and also eliciting increased production of pro-inflammatory chemokines from cervicovaginal epithelial cells (14
). Our data are consistent with this prediction and show that (i) SEVI enhances the ability of HIV-1 virions to elicit IL-8 and MIP-3α from A2En endocervical cells and (ii) this can be inhibited by BTA-EG6
. This is important because it suggests that BTA-EG6
and related compounds may have the potential not only to reduce the efficiency of HIV-1 infection of target cells but also to reduce the level of target cell recruitment to virus-exposed genital mucosal tissue.
Our results show that BTA-EG6
effectively prevents semen-mediated enhancement of HIV infectivity, suggesting that this activity of semen can be targeted by specifically inhibiting the SEVI fibrils. BTA-EG6
did not inhibit other properties of semen, such as the ability to elicit pro-inflammatory chemokines (13
), further reinforcing the idea that this small molecule specifically targets the fibrils present in semen. These data not only suggest that BTA-EG6
may be an effective microbicide target but also offer proof of principle support for the concept that screening amyloid- or SEVI-binding molecules may allow identification of additional anti-SEVI microbicide candidates. In the case of BTA-EG6
, it is likely that the thioflavin-T analog allows it to intercalate into the SEVI fibrils, whereas the hexa(ethylene glycol) moiety extends and inhibits the interactions of SEVI with cells and virions (6
). Oligomeric derivatives of BTA-EG6
and related compounds may prove even more effective due to increased avidity and affinity, as may amyloid binders with modified functional groups. Studies to address these hypotheses are presently underway.
Some previous microbicide candidates have not fared well in clinical trials, frequently due to pro-inflammatory properties that damage the cervical mucosa and drive the recruitment of HIV target cells. An instructive example is the nonionic surfactant, nonoxynol-9. Nonoxynol-9 showed strong antiviral activity in preclinical trials but failed to protect against virus transmission in human clinical trials, and in fact, increased the risk of HIV-1 infection by almost 2-fold in women who used the agent several times a day (20
). This infection-enhancing activity has been attributed to toxic and pro-inflammatory effects on female reproductive tissue, which increase susceptibility to virus transmission (22
). Therefore, it is important to consider toxicity when evaluating candidate microbicides. We have found that BTA-EG6
is not toxic to a variety of cervical cells and that it does not drive the expression of pro-inflammatory cytokines and chemokines that have been shown to be important in recruiting HIV target cells.
An additional consideration for many candidate microbicides is that their properties can, in some cases, be negatively impacted by the presence of semen. The anionic polymer PRO-2000 also showed robust activity in in vitro
studies and preclinical models (23
) but failed to demonstrate a protective effect against HIV-1 transmission in the phase III Microbicide Development Program (MDP) 301 trial.3
Reasons for the futility of PRO-2000 remain unclear but may include inhibition of its antiviral activity by seminal plasma (27
); this may contribute to its decreased effectiveness in postcoital cervicovaginal fluid (28
). In our experiments, the presence of semen had no effect on the microbicidal activity of BTA-EG6
It is important to compare the properties of the anti-SEVI small molecules described here with those of previously identified inhibitors of SEVI-enhanced HIV infection activity. Other previously described SEVI inhibitors, such as polyanionic compounds (3
) and the heparin antagonist surfen (29
), block the actions of SEVI at least in part due to simple electrostatic interactions with the cationic fibrils. Given recent experience with the polyanionic agent PRO-20003
and the presence of small cationic peptides in both semen and cervicovaginal fluid (30
), this raises concerns. In contrast, amyloid-binding small molecules, such as BTA-EG6
, do not rely on electrostatic properties to bind to SEVI and are therefore expected to offer a new class of SEVI inhibitors whose effects are more specifically targeted at the fibrils themselves. This is expected to reduce potential for off-target effects and increase potential for effectiveness in an in vivo
setting. Finally, current microbicide candidates target the HIV virus itself (32
), whereas the inhibitor described here does not. We propose that because semen is the vector in the vast majority of transmitted HIV-1 infections, it is reasonable to consider that the addition of a SEVI inhibitor to microbicide formulations might improve the efficacy of antiviral microbicides.