Toxicity and in vitro activity of HIV-1 latency-reversing agents in primary CNS cells.

Despite the success of combination antiretroviral therapy (cART), HIV persists in long lived latently infected cells in the blood and tissue, and treatment is required lifelong. Recent clinical studies have trialed latency-reversing agents (LRA) as a method to eliminate latently infected cells; however, the effects of LRA on the central nervous system (CNS), a well-known site of virus persistence on cART, are unknown. In this study, we evaluated the toxicity and potency of a panel of commonly used and well-known LRA (panobinostat, romidepsin, vorinostat, chaetocin, disulfiram, hexamethylene bisacetamide [HMBA], and JQ-1) in primary fetal astrocytes (PFA) as well as monocyte-derived macrophages as a cellular model for brain perivascular macrophages. We show that most LRA are non-toxic in these cells at therapeutic concentrations. Additionally, romidepsin, JQ-1, and panobinostat were the most potent at inducing viral transcription, with greater magnitude observed in PFA. In contrast, vorinostat, chaetocin, disulfiram, and HMBA all demonstrated little or no induction of viral transcription. Together, these data suggest that some LRA could potentially activate transcription in latently infected cells in the CNS. We recommend that future trials of LRA also examine the effects of these agents on the CNS via examination of cerebrospinal fluid.

Hyperimmune bovine colostrum as a low-cost, large-scale source of antibodies with broad neutralizing activity for HIV-1 envelope with potential use in microbicides.

Bovine colostrum (first milk) contains very high concentrations of IgG, and on average 1 kg (500 g/liter) of IgG can be harvested from each immunized cow immediately after calving. We used a modified vaccination strategy together with established production systems from the dairy food industry for the large-scale manufacture of broadly neutralizing HIV-1 IgG. This approach provides a low-cost mucosal HIV preventive agent potentially suitable for a topical microbicide. Four cows were vaccinated pre- and/or postconception with recombinant HIV-1 gp140 envelope (Env) oligomers of clade B or A, B, and C. Colostrum and purified colostrum IgG were assessed for cross-clade binding and neutralization against a panel of 27 Env-pseudotyped reporter viruses. Vaccination elicited high anti-gp140 IgG titers in serum and colostrum with reciprocal endpoint titers of up to 1 × 10(5). While nonimmune colostrum showed some intrinsic neutralizing activity, colostrum from 2 cows receiving a longer-duration vaccination regimen demonstrated broad HIV-1-neutralizing activity. Colostrum-purified polyclonal IgG retained gp140 reactivity and neutralization activity and blocked the binding of the b12 monoclonal antibody to gp140, showing specificity for the CD4 binding site. Colostrum-derived anti-HIV antibodies offer a cost-effective option for preparing the substantial quantities of broadly neutralizing antibodies that would be needed in a low-cost topical combination HIV-1 microbicide.

HIV latency can be established in proliferating and nonproliferating resting CD4+ T cells in vitro: implications for latency reversal.

OBJECTIVE: To determine whether latency can be established and reversed in both proliferating and nonproliferating CD4+ T cells in the same model in vitro. METHODS: Activated CD4+ T cells were infected with either a nonreplication competent, luciferase reporter virus or wild-type full-length enhanced green fluorescent protein (EGFP) reporter virus and cultured for 12 days. The cells were then sorted by flow cytometry to obtain two distinct T-cell populations that did not express the T-cell activation markers, CD69, CD25 and human leukocyte antigen (HLA)-DR: CD69CD25HLA-DR small cells (nonblasts) that had not proliferated in vitro following mitogen stimulation and CD69CD25HLA-DR large cells (which we here call transitional blasts) that had proliferated. The cells were then reactivated with latency-reversing agents and either luciferase or EGFP quantified. RESULTS: Inducible luciferase expression, consistent with latent infection, was observed in nonblasts and transitional blasts following stimulation with either phorbol-myristate-acetate/phytohemagglutinin (3.8 ±â€Š1 and 2.9 ±â€Š0.5 fold above dimethyl sulfoxide, respectively) or romidepsin (2.1 ±â€Š0.6 and 1.8 ±â€Š0.2 fold above dimethyl sulfoxide, respectively). Constitutive expression of luciferase was higher in transitional blasts compared with nonblasts. Using wild-type full-length EGFP reporter virus, inducible virus was observed in nonblasts but not in transitional blasts. No significant difference was observed in the response to latency-reversing agents in either nonblasts or transitional blasts. CONCLUSION: HIV latency can be established in vitro in resting T cells that have not proliferated (nonblasts) and blasts that have proliferated (transitional blasts). This model could potentially be used to assess new strategies to eliminate latency.