In vitro Evidence That Combination Therapy With CD16-Bearing NK-92 Cells and FDA-Approved Alefacept Can Selectively Target the Latent HIV Reservoir in CD4+ CD2hi Memory T Cells.

Elimination of the latent HIV reservoir remains the biggest hurdle to achieve HIV cure. In order to specifically eliminate HIV infected cells they must be distinguishable from uninfected cells. CD2 was recently identified as a potential marker enriched in the HIV-1 reservoir on CD4+ T cells, the largest, longest-lived and best-characterized constituent of the HIV reservoir. We previously proposed to repurpose FDA-approved alefacept, a humanized α-CD2 fusion protein, to reduce the HIV reservoir in CD2hi CD4+ memory T cells. Here, we show the first evidence that alefacept can specifically target and reduce CD2hi HIV infected cells in vitro. We explore a variety of natural killer (NK) cells as mediators of antibody-dependent cell-mediated cytotoxicity (ADCC) including primary NK cells, expanded NK cells as well as the CD16 transduced NK-92 cell line which is currently under study in clinical trials as a treatment for cancer. We demonstrate that CD16.NK-92 has a natural preference to kill CD2hi CD45RA- memory T cells, specifically CD45RA- CD27+ central memory/transitional memory (TCM/TM) subset in both healthy and HIV+ patient samples as well as to reduce HIV DNA from HIV+ samples from donors well controlled on antiretroviral therapy. Lastly, alefacept can combine with CD16.NK-92 to decrease HIV DNA in some patient samples and thus may yield value as part of a strategy toward sustained HIV remission.

Dimerization of the Pseudomonas aeruginosa translocator chaperone PcrH is required for stability, not function.

Type III secretion systems rely on hydrophobic translocator proteins that form a pore in the host cell membrane to deliver effector proteins into targeted host cells. These translocator proteins are stabilized in the cytoplasm and targeted for export with the help of specific chaperone proteins. In Pseudomonas aeruginosa, the chaperone of the pore-forming translocator proteins is PcrH. Although all translocator chaperones dimerize, the location of the dimerization interface is in dispute. Moreover, it has been reported that interfering with dimerization interferes with chaperone function. However, binding of P. aeruginosa chaperone PcrH to its cognate secretion substrate, PopD, results in dissociation of the PcrH dimer in vitro, arguing that dimerization of PcrH is likely not important for substrate binding or targeting translocators for export. We demonstrate that PcrH dimerization occurs in vivo in P. aeruginosa and used a genetic screen to identify a dimerization mutant of PcrH. The mutant protein is fully functional in that it can both stabilize PopB and PopD in the cytoplasm and promote their export via the type III secretion system. The location of the mutation suggests that the dimerization interface of PcrH mirrors that of the Yersinia homolog SycD and not the dimerization interface that had previously been reported for PcrH based on crystallographic evidence. Finally, we present data that the dimerization mutant of PcrH is less stable than the wild-type protein in P. aeruginosa, suggesting that the function of dimerization is stabilization of PcrH in the absence of its cognate cargo.

A translocator-specific export signal establishes the translocator-effector secretion hierarchy that is important for type III secretion system function.

Type III secretion systems are used by many Gram-negative pathogens to directly deliver effector proteins into the cytoplasm of host cells. To accomplish this, bacteria secrete translocator proteins that form a pore in the host-cell membrane through which the effector proteins are then introduced into the host cell. Evidence from multiple systems indicates that the pore-forming translocator proteins are exported before effectors, but how this secretion hierarchy is established is unclear. Here we used the Pseudomonas aeruginosa translocator protein PopD as a model to identify its export signals. The N-terminal secretion signal and chaperone, PcrH, are required for export under all conditions. Two novel signals in PopD, one proximal to the chaperone binding site and one at the very C-terminus of the protein, are required for export of PopD before effector proteins. These novel export signals establish the translocator-effector secretion hierarchy, which in turn, is critical for the delivery of effectors into host cells.