Translational Research - A Basic Science Perspective Translational research: translates basic science discoveries into clinical applications, and/or uses clinical observations to generate new research topics. Focus is on the integration of activities from bench to bedside.
Significance for vertical transmission. Vertical transmission of HIV results in the infection of thousands of children every year. Although vertical transmission frequently occurs during birth, a substantial proportion also occurs in utero. The presence of DC-SIGN and DC-SIGNR on specific cell types in the placenta (both maternal and fetal sides of the circulation) raises the possibility that these attachment factors could impact this process. We do not plan to directly test this possibility in vivo at the present time, concentrating instead on the potential role of DC-SIGN in sexual transmission as described above. However, our in vitro studies and detailed examination of DC-SIGN and DC-SIGNR expression may help guide future in vivo experiments or determine if they are even warranted. For example, which specific cell types in the placenta express DC-SIGN and DC-SIGNR (Specific Aim #4)? At what levels are these attachment factors expressed (Specific Aim #4); and are there differences in how viruses interact with these attachment factors (Specific Aim #2)? If there are differences, do these correlate with specific virus types (Specific Aim #2)? We feel that the studies proposed in this grant will provide important baseline information that will be needed to consider this question in the future.
We have found that there is considerable variability in how avidly Env proteins from different virus strains bind to their respective coreceptors. While we have rigorously examined binding constants for only a handful of Env proteins, it is clear that binding affinities can range from at least 4 nM to approximately 500 nM - a variance of two orders of magnitude . Do differences in how Env proteins interact with their coreceptors have any impact on sensitivity to entry inhibitors? A related question concerns mechanisms by which virus can acquire resistance to entry inhibitors - a very real concern given that entry inhibitors are now in clinical trials. Is there any cost associated with resistance to entry inhibitors, and can entry inhibitors be used in various combinations to limit virus evolution? These questions will be addressed in Specific Aim #3. In our preliminary studies, we have begun to examine how differences in Env-coreceptor interactions impact sensitivity to T20 and how viruses develop resistance to the CCR5 antagonist TAK779. We have also begun to survey primary virus strains for their sensitivity to a panel of entry inhibitors used singly and in combination. Finally, we have obtained sufficient quantities of T20 and T1249 (a more potent derivative of T20) from Trimeris, AMD3100 from Anormed, and TAK779 for the experiments described in Specific Aim #3.
D1a. Selection of mutations and choice of Env. Initially, we will introduce 13 single amino acid substitutions in the bridging sheet region of IIIB. As shown in Section C3, we have already gotten a good start on this project, and the specific mutations are listed in Table 1. A potential problem with our approach is that the HxB gp120 binds to CXCR4 with poor affinity . By contrast, HxB-V3-BaL gp120 binds well to CCR5. Thus, while it is relatively trivial to study the impact of these mutations on CXCR4-dependent membrane fusion activity, either in the context of cell-cell fusion or virus entry assays, it is more difficult to measure their impact on gp120-CXCR4 binding. As discussed below, we should be able to accomplish this goal. However, it may prove desirable to place some of these mutations into an X4 Env protein that binds to CXCR4 with high affinity, though we have not yet identified such a protein, at least for HIV-1. This is discussed in Section D1d.