Anti-HIV microbicides are being investigated in clinical trials and understanding how promising strategies work, coincident with demonstrating efficacy in vivo, is central to advancing new generation microbicides. We evaluated Carraguard® and a new generation Carraguard-based formulation containing the non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 (PC-817). Since dendritic cells (DCs) are believed to be important in HIV transmission, the formulations were tested for the ability to limit DC-driven infection in vitro versus vaginal infection of macaques with RT-SHIV (SIVmac239 bearing HIV reverse transcriptase). Carraguard showed limited activity against cell-free and mature DC-driven RT-SHIV infections and, surprisingly, low doses of Carraguard enhanced infection. However, nanomolar amounts of MIV-150 overcame enhancement and blocked DC-transmitted infection. In contrast, Carraguard impeded infection of immature DCs coincident with DC maturation. Despite this variable activity in vitro, Carraguard and PC-817 prevented vaginal transmission of RT-SHIV when applied 30 min prior to challenge. PC-817 appeared no more effective than Carraguard in vivo, due to the limited activity of a single dose of MIV-150 and the dominant barrier effect of Carraguard. However, 3 doses of MIV-150 in placebo gel at and around challenge limited vaginal infection, demonstrating the potential activity of a topically applied NNRTI. These data demonstrate discordant observations when comparing in vitro and in vivo efficacy of Carraguard-based microbicides, highlighting the difficulties in testing putative anti-viral strategies in vitro to predict in vivo activity. This work also underscores the potential of Carraguard-based formulations for the delivery of anti-viral drugs to prevent vaginal HIV infection.

BackgroundHerpes simplex virus type-2 (HSV-2) infection enhances the transmission and acquisition of human immunodeficiency virus (HIV). This occurs in symptomatic and asymptomatic stages of HSV-2 infection, suggesting that obvious herpetic lesions are not required to increase HIV spread. An animal model to investigate the underlying causes of the synergistic action of the two viruses and where preventative strategies can be tested under such complex physiological conditions is currently unavailable.Methodology/Principal FindingsWe set out to establish a rhesus macaque model in which HSV-2 infection increases the susceptibility to vaginal infection with a model immunodeficiency virus (simian-human immunodeficiency virus, SHIV-RT), and to more stringently test promising microbicides. HSV-2 exposure significantly increased the frequency of vaginal SHIV-RT infection (n = 6). Although cervical lesions were detected in only ∼10% of the animals, long term HSV-2 DNA shedding was detected (in 50% of animals followed for 2 years). Vaginal HSV-2 exposure elicited local cytokine/chemokine (n = 12) and systemic low-level HSV-2-specific adaptive responses in all animals (n = 8), involving CD4+ and CD8+ HSV-specific T cells (n = 5). Local cytokine/chemokine responses were lower in co-infected animals, while simian immunodeficiency virus (SIV)-specific adaptive responses were comparable in naïve and HSV-2-infected animals (n = 6). Despite the increased frequency of SHIV-RT infection, a new generation microbicide gel, comprised of Carraguard® and a non-nucleoside reverse transcriptase inhibitor MIV-150 (PC-817), blocked vaginal SHIV-RT infection in HSV-2-exposed animals (n = 8), just as in naïve animals.Conclusions/SignificanceWe established a unique HSV-2 macaque model that will likely facilitate research to define how HSV-2 increases HIV transmission, and enable more rigorous evaluation of candidate anti-viral approaches in vivo.

Aureusimines have been identified as potential virulence factors in Staphylococcus aureus. These pyrazinone secondary metabolites are produced by a nonribosomal peptide synthetase (NRPS) annotated as AusA. We report the overproduction of AusA as a 277 kDa soluble protein with the bimodular architecture A1–T1–C–A2–T2–R. The substrate specificity of each adenylation (A) domain was initially probed using an ATP–pyrophosphate exchange assay with A-domain selective bisubstrate inhibitors to chemically knock-out each companion A-domain. The activity of AusA was then reconstituted in vitro and shown to produce all naturally occurring aureusimines and non-natural pyrazinone products with kcat values ranging from 0.4 to 1.3 min−1. Steady-state kinetic parameters were determined for all substrates and cofactors providing the first comprehensive steady-state characterization of a NRPS employing a product formation assay. The KM values for the amino acids were up to 60-fold lower with the product formation assay versus the ATP–pyrophosphate exchange assay, most commonly used to assess A-domain substrate specificity. The C-terminal reductase (R) domain catalyzes reductive release of the dipeptidyl intermediate leading to formation of an amino aldehyde that cyclizes to a dihydropyrazinone. We show oxidation to the final pyrazinone heterocycle is spontaneous. The activity and specificity of the R-domain was independently investigated using a NADPH consumption assay. AusA is a minimal autonomous two-module NRPS that represents an excellent model system for further kinetic and structural characterization.

A simple steady-state kinetic high-throughput assay was developed for the salicylate synthase MbtI from Mycobacterium tuberculosis, which catalyzes the first committed step of mycobactin biosynthesis. The mycobactins are small-molecule iron chelators produced by M. tuberculosis, and their biosynthesis has been identified as a promising target for the development of new antitubercular agents. The assay was miniaturized to a 384-well plate format and high-throughput screening was performed at the National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases (NSRB). Three classes of compounds were identified comprising the benzisothiazolones (class I), diarylsulfones (class II), and benzimidazole-2-thiones (class III). Each of these compound series was further pursued to investigate their biochemical mechanism and structure-activity relationships. Benzimidazole-2-thione 4 emerged as the most promising inhibitor owing to its potent reversible inhibition.

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