Bridging the Gap between Preclinical and Clinical Microbicide Trials: Blind Evaluation of Candidate Gels in Murine Models of Efficacy and Safety

Despite significant protection in preclinical studies, cellulose sulfate (CS) failed to protect women against HIV-1/2 and was associated with a trend toward increased HIV-1 acquisition in one of the clinical trials. These results highlight the need for preclinical tests more predictive of clinical outcomes. The objective of this study was to test coded vaginal gels, including CS, in murine models of safety and efficacy to determine the models' utility for evaluating future products.Four coded formulations, including 6% CS, 2% PRO 2000 and two placebo gels, were administered intravaginally to medroxyprogesterone-treated mice and their ability to prevent genital herpes (efficacy) or to alter the susceptibility to low dose HSV challenge (safety) was determined. Nonoyxnol-9 served as a positive toxicity control.CS and PRO 2000 significantly protected mice from genital herpes following infection with a laboratory or clinical isolate of HSV-2 introduced in buffer (p<0.001). However, protection was reduced when virus was introduced in seminal plasma. Moreover, mice were significantly more susceptible to infection with low doses of HSV-2 when challenged 12 h after the 7th daily dose of CS or nonoxynol-9 (p<0.05). The increased susceptibility was associated with alterations in epithelial architecture.CS prevented genital herpes when present at the time of viral challenge, but increased the rate of infection when gel was applied daily for 7 days with a vaginal wash prior to viral inoculation. The findings presumably reflect altered epithelial architecture, which may have contributed to the trend towards increased HIV observed clinically

HSV Usurps Eukaryotic Initiation Factor 3 Subunit M for Viral Protein Translation: Novel Prevention Target

Prevention of genital herpes is a global health priority. B5, a recently identified ubiquitous human protein, was proposed as a candidate HSV entry receptor. The current studies explored its role in HSV infection. Viral plaque formation was reduced by ∼90% in human cells transfected with small interfering RNA targeting B5 or nectin-1, an established entry receptor. However, the mechanisms were distinct. Silencing of nectin-1 prevented intracellular delivery of viral capsids, nuclear transport of a viral tegument protein, and release of calcium stores required for entry. In contrast, B5 silencing had no effect on these markers of entry, but inhibited viral protein translation. Specifically, viral immediate early genes, ICP0 and ICP4, were transcribed, polyadenylated and transported from the nucleus to the cytoplasm, but the viral transcripts did not associate with ribosomes or polysomes in B5-silenced cells. In contrast, immediate early gene viral transcripts were detected in polysome fractions isolated from control cells. These findings are consistent with sequencing studies demonstrating that B5 is eukaryotic initiation factor 3 subunit m (eIF3m). Although B5 silencing altered the polysome profile of cells, silencing had little effect on cellular RNA or protein expression and was not cytotoxic, suggesting that this subunit is not essential for host cellular protein synthesis. Together these results demonstrate that B5 plays a major role in the initiation of HSV protein translation and could provide a novel target for strategies to prevent primary and recurrent herpetic disease

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure &lt; 100 mmHg (n = 1127), estimated glomerular filtration rate &lt; 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Silencing of B5 blocks translation, but not transcription, of viral proteins.

<p>Cells were transfected with siHVEM or siB5 and then infected with HSV-2(G) and 4 h pi, RNA was extracted and analyzed by qRT-PCR for viral gene expression. RNA was also extracted from non-transfected CaSki cells that had been infected with HSV-2(G) in the presence of cycloheximide. Results are presented as RNA expressed in siB5 transfected cells or in cells infected in the presence of cycloheximide as a percentage of expression in siHVEM transfected cells and are mean ± sd obtained from 3 independent experiments.</p

Viral RNA and immediate early proteins associate with B5 in RNA-protein complexes.

<p>(A) Cells were infected with HSV-2(G) and 3, 5, or 7 h pi, the cells were treated with 0.04% of methanol-free formaldehyde to cross-link RNA-protein complexes. The complexes were immunoprecipitated with a polyclonal rabbit anti-B5. and RNA was extracted from the supernatant and pellet and analyzed for the expression of viral transcripts and RPLPO. Results are shown for the time pi at which peak expression of viral transcripts was detected (3 h for ICP4, 5 h for ICP0 and 7 h for gB) and are presented as the ratio of viral transcript/RPLPO in each fraction to the viral transcipts/RPLPO in the total RNA-protein complex from infected cells prior to immunoprecipitation (input). Expression of RPLPO is presented as the ratio of RPLPO in each fraction to RPLPO in the total RNA-protein complex from uninfected cells prior to immunoprecipitation (input). (B) The supernatant and pellet from the immunoprecipitated complexes isolated at 0, 3, 5, and 7 h pi were evaluated for the expression of ICP4 (HSV-2), gB (HSV-2), ICP0 (HSV-1) or RPLPO by Western blots. Blots are representative of results obtained in at least 3 independent experiments.</p

Silencing of B5 and nectin-1 reduces HSV infection.

<p>(A). CaSki cells were transfected with siHVEM (control), B5 or nectin-1-specific pools of siRNA and 48 h post-transfection, immunoblots of cell lysates were prepared and probed with anti-B5 polyclonal Ab or anti-nectin mAb and then stripped and probed for β-actin expression. (B) Western blots were scanned for protein expression (odu). In parallel studies, RNA was extracted from transfected cells and analyzed by qRT-PCR for expression of B5 and nectin. Results are presented as protein or RNA expression as a percentage of cells transfected with siHVEM and are means ± sd obtained from 3 different experiments. (C) The transfected cells were infected with HSV-2(G) or HSV-1(KOS) (48 h post-transfection) and the ability to support viral plaque formation determined by counting plaques after 48 h incubation by immunostaining. Results are presented as pfu/well as a percentage of pfu/well formed in cells transfected with non-specific control siRNA and are means + sd from 2 independent experiments conducted in quadruplicate. (D) Cells were transfected independently with each siB5, with the pool of three siRNAs or with siHVEM (control) and 48 h post-transfection immunoblots of cell lysates were prepared and probed with anti-B5 polyclonal Ab and β-actin and relative expression determined after scanning the blots. Results are presented as odu relative to cells transfected with siHVEM. In parallel, the transfected cells were infected with HSV-2(G) and the ability to support viral plaque formation determined by counting plaques after 48 h incubation by immunostaining. Results are presented as pfu/well as percentage of pfu/well formed in cells transfected with siHVEM and are means ± sd from a representative experiment conducted in duplicate.</p

Silencing of B5 blocks viral protein expression.

<p>(A). CaSki cells were transfected with siB5 or siHVEM and then 48 h later, synchronously infected with HSV-2(G) and nuclear extracts harvested at the indicated times and evaluated for VP16. Blots are representative of results obtained in 3 independent experiments. (B). Transfected cells were infected with HSV-1(KOS) and 4 h pi, cellular lysates harvested and analyzed for the expression of ICP4, ICP0 and ICP27. Blots were scanned and the relative expression of viral proteins by odu is presented as a percentage of expression in cells transfected with siHVEM obtained in 3 independent experiments (right).</p

B5 silencing has more modest effects on HIV or VSV infection.

<p>(A) Jurkat-TAT-CCR5 cells were transfected with siHVEM (control) or B5 specific siRNA and 48 h post-transfection, immunoblots of cell lysates were prepared and probed with anti-B5 polyclonal Ab (upper panel). Transfected cells were infected with HSV-1(KOS) and 4 h pi, cell lysates prepared and analyzed for ICP0 expression (middle panel) and β-actin as a loading control (lower panel). In parallel, the transfected cells were infected with HIV-1(BaL) and culture supernatants harvested 5 days pi and analyzed for p24 expression (right). (B) CaSki cells were transfected with HVEM or B5 specific siRNA and 48 h post-transfection immunoblots of cell lysates were prepared and probed with anti-B5 polyclonal Ab (upper) and β-actin as a loading control (lower panel). The transfected cells were infected with HSV-2(G) or VSV and plaques counted 48 h pi; results are means ± sd obtained from two independent experiments conducted in duplicate.</p

Acyclovir and B5 silencing inhibit HSV infection and silencing has little impact on host cell protein synthesis.

<p>(A). Cells were either transfected with HVEM or B5 specific siRNA or not transfected, and then infected with serial dilutions of HSV-2(G) (0.001–1000 pfu/cell) in the absence or presence of acyclovir (ACV) (100µg/ml) and 48 h pi, cells were fixed and viral plaques counted. Only wells with 20–150 pfu were counted to determine the viral titer (pfu/ml). (B). Transfected cells were metabolically labeled 72 h post-transfection and analyzed by gel electrophoresis followed by autoradiography. Results are representative of 3 independent experiments (left). Immunoblot of cells 72 h post-transfection probed with antibodies to B5 and β-actin (right).</p

B5 silencing does not block polyadenylation or nuclear export of RNA.

<p>(A) Transfected cells were infected with HSV-2(G) (moi 3 pfu/cell) and 3 h pi, cells were harvested and nuclear and cytoplasmic fractions isolated and Western blots were prepared and probed for golgin97, histone-1, B5 and ICP4. (B) Polyadenylated mRNA was isolated from the total RNA and from the cytoplasmic and nuclear fractions and then analyzed for ICP4 by qRT-PCR. Results are presented as the ratio of ICP4/RPLPO in each fraction (cytoplasmic or nuclear) to ICP4/RPLPO in the unfractionated total polyA RNA and are representative of 3 independent experiments.</p