HIV-1 Nef binds the DOCK2-ELMO1 complex to activate rac and inhibit lymphocyte chemotaxis

The infectious cycle of primate lentiviruses is intimately linked to interactions between cells of the immune system. Nef, a potent virulence factor, alters cellular environments to increase lentiviral replication in the host, yet the mechanisms underlying these effects have remained elusive. Since Nef likely functions as an adaptor protein, we exploited a proteomic approach to directly identify molecules that Nef targets to subvert the signaling machinery in T cells. We purified to near homogeneity a major Nef-associated protein complex from T cells and identified by mass spectroscopy its subunits as DOCK2–ELMO1, a key activator of Rac in antigen- and chemokine-initiated signaling pathways, and Rac. We show that Nef activates Rac in T cell lines and in primary T cells following infection with HIV-1 in the absence of antigenic stimuli. Nef activates Rac by binding the DOCK2–ELMO1 complex, and this interaction is linked to the abilities of Nef to inhibit chemotaxis and promote T cell activation. Our data indicate that Nef targets a critical switch that regulates Rac GTPases downstream of chemokine- and antigen-initiated signaling pathways. This interaction enables Nef to influence multiple aspects of T cell function and thus provides an important mechanism by which Nef impacts pathogenesis by primate lentiviruses.     

Randomized, controlled trial of therapy interruption in chronic HIV-1 infection

Background

Approaches to limiting exposure to antiretroviral therapy (ART) drugs are an active area of HIV therapy research. Here we present longitudinal follow-up of a randomized, open-label, single-center study of the immune, viral, and safety outcomes of structured therapy interruptions (TIs) in patients with chronically suppressed HIV-1 infection as compared to equal follow-up of patients on continuous therapy and including a final therapy interruption in both arms.

Methods and Findings

Forty-two chronically HIV-infected patients on suppressive ART with CD4 counts higher than 400 were randomized 1:1 to either (1) three successive fixed TIs of 2, 4, and 6 wk, with intervening resumption of therapy with resuppression for 4 wk before subsequent interruption, or (2) 40 wk of continuous therapy, with a final open-ended TI in both treatment groups. Main outcome was analysis of the time to viral rebound (>5,000 copies/ml) during the open-ended TI. Secondary outcomes included study-defined safety criteria, viral resistance, therapy failure, and retention of immune reconstitution.There was no difference between the groups in time to viral rebound during the open-ended TI (continuous therapy/single TI, median [interquartile range] = 4 [1–8] wk, n = 21; repeated TI, median [interquartile range] = 5 [4–8] wk, n = 21; p = 0.36). No differences in study-related adverse events, viral set point at 12 or 20 wk of open-ended interruption, viral resistance or therapy failure, retention of CD4 T cell numbers on ART, or retention of lymphoproliferative recall antigen responses were noted between groups. Importantly, resistance detected shortly after initial viremia following the open-ended TI did not result in a lack of resuppression to less than 50 copies/ml after reinitiation of the same drug regimen.

Conclusion

Cycles of 2- to 6-wk time-fixed TIs in patients with suppressed HIV infection failed to confer a clinically significant benefit with regard to viral suppression off ART. Also, secondary analysis showed no difference between the two strategies in terms of safety, retention of immune reconstitution, and clinical therapy failure. Based on these findings, we suggest that further clinical research on the long-term consequences of TI strategies to decrease drug exposure is warranted.     

A randomised, double-blind, controlled vaccine efficacy trial of DNA/MVA ME-TRAP against malaria infection in Gambian adults

Background

Many malaria vaccines are currently in development, although very few have been evaluated for efficacy in the field. Plasmodium falciparum multiple epitope (ME)– thrombospondin-related adhesion protein (TRAP) candidate vaccines are designed to potently induce effector T cells and so are a departure from earlier malaria vaccines evaluated in the field in terms of their mechanism of action. ME-TRAP vaccines encode a polyepitope string and the TRAP sporozoite antigen. Two vaccine vectors encoding ME-TRAP, plasmid DNA and modified vaccinia virus Ankara (MVA), when used sequentially in a prime-boost immunisation regime, induce high frequencies of effector T cells and partial protection, manifest as delay in time to parasitaemia, in a clinical challenge model.

Methods and Findings

A total of 372 Gambian men aged 15–45 y were randomised to receive either DNA ME-TRAP followed by MVA ME-TRAP or rabies vaccine (control). Of these men, 296 received three doses of vaccine timed to coincide with the beginning of the transmission season (141 in the DNA/MVA group and 155 in the rabies group) and were followed up. Volunteers were given sulphadoxine/pyrimethamine 2 wk before the final vaccination. Blood smears were collected weekly for 11 wk and whenever a volunteer developed symptoms compatible with malaria during the transmission season. The primary endpoint was time to first infection with asexual P. falciparum. Analysis was per protocol.DNA ME-TRAP and MVA ME-TRAP were safe and well-tolerated. Effector T cell responses to a non-vaccine strain of TRAP were 50-fold higher postvaccination in the malaria vaccine group than in the rabies vaccine group. Vaccine efficacy, adjusted for confounding factors, was 10.3% (95% confidence interval, −22% to +34%; p = 0.49). Incidence of malaria infection decreased with increasing age and was associated with ethnicity.

Conclusions

DNA/MVA heterologous prime-boost vaccination is safe and highly immunogenic for effector T cell induction in a malaria-endemic area. But despite having produced a substantial reduction in liver-stage parasites in challenge studies of non-immune volunteers, this first generation T cell–inducing vaccine was ineffective at reducing the natural infection rate in semi-immune African adults.     

The proliferation associated nuclear element (PANE1) is conserved between mammals and fish and preferentially expressed in activated lymphoid cells

The proliferation associated nuclear element (PANE1) had been identified in a screen for genes activated in mouse mammary epithelium transformed by stabilized beta-catenin. We have now cloned the human and zebrafish orthologs, analyzed their expression and expressed them ectopically in tissue culture cell lines. PANE1 consists of 180 amino acids and displays 38% conservation between man and zebrafish. Expression of the human PANE1 gene was detected preferentially in immune cells including leukemias and lymphomas, tumor tissues and tumor derived cell lines. In B- and T-cells PANE1 RNA was only detected after the respective cell types were activated either in vivo or in vitro.     

Use and optimization of a dual-flowrate loading strategy to maximize throughput in protein-a affinity chromatography

The effect of an alternate strategy employing two different flowrates during loading was explored as a means of increasing system productivity in Protein-A chromatography. The effect of such a loading strategy was evaluated using a chromatographic model that was able to accurately predict experimental breakthrough curves for this Protein-A system. A gradient-based optimization routine is carried out to establish the optimal loading conditions (initial and final flowrates and switching time). The two-step loading strategy (using a higher flowrate during the initial stages followed by a lower flowrate) was evaluated for an Fc-fusion protein and was found to result in significant improvements in process throughput. In an extension of this optimization routine, dynamic loading capacity and productivity were simultaneously optimized using a weighted objective function, and this result was compared to that obtained with the single flowrate. Again, the dual-flowrate strategy was found to be superior.     

Maximizing recovery of native protein from aggregates by optimizing pressure treatment

Recovering native protein from aggregates is a common obstacle in the production of recombinant proteins. Recent reports have shown that hydrostatic pressure is an attractive alternative to traditional denature-and-dilute techniques, both in terms of yield and process simplicity. To determine the effect of process variables, we subjected tailspike aggregates to a variety of pressure-treatment conditions. Maximum native tailspike yields were obtained with only short pressure incubations (<5 min) at 240 MPa. However, some tailspike aggregates were resistant to pressure, despite multiple cycles of pressure. Extending the postpressure incubation time to 4 days improved the yield of native protein from aggregates from 19.4 +/- 0.9 to 47.4 +/- 19.6 microg/mL (approximately 78% yield of native trimer from nonaggregate material). The nearly exclusive conversion of monomer to trimer over the time scale of days, when combined with previous kinetic data, allows for the identification of three postpressure kinetic phases: a rapid phase consisting of structured dimer conversion to trimer (30 min), an intermediate phase consisting of monomer conversion to aggregate (100 min), and a slow phase consisting of conversion of monomer to trimer (days). Optimizing the production of structured dimer can yield the highest level of folded protein. Typical refolding additives, such as glycerol, or low-temperature incubation did not improve yields.     

Effects of short-term chemical ablation of the GIP receptor on insulin secretion, islet morphology and glucose homeostasis in mice

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone secreted by endocrine K-cells in response to nutrient absorption. In this study we have utilized a specific and enzymatically stable GIP receptor antagonist, (Pro3)GIP, to evaluate the contribution of endogenous GIP to insulin secretion and glucose homeostasis in mice. Daily injection of (Pro3)GIP (25 nmol/kg body weight) for 11 days had no effect on food intake or body weight. Non-fasting plasma glucose concentrations were significantly raised (p<0.05) by day 11, while plasma insulin concentrations were not significantly different from saline treated controls. After 11 days, intraperitoneal glucose tolerance was significantly impaired in the (Pro3)GIP treated mice compared to control (p<0.01). Glucose-mediated insulin secretion was not significantly different between the two groups. Insulin sensitivity of 11-day (Pro3)GIP treated mice was slightly impaired 60 min post injection compared with controls. Following a 15 min refeeding period in 18 h fasted mice, food intake was not significantly different in (Pro3)GIP treated mice and controls. However, (Pro3)GIP treated mice displayed significantly elevated plasma glucose levels 30 and 60 min post feeding (p<0.05, in both cases). Postprandial insulin secretion was not significantly different and no changes in pancreatic insulin content or islet morphology were observed in (Pro3)GIP treated mice. The observed biological effects of (Pro3)GIP were reversed following cessation of treatment for 9 days. These data indicate that ablation of GIP signaling causes a readily reversible glucose intolerance without appreciable change of insulin secretion.