HIV This Week

 Scaleable manufacture of HIV-1 entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component. O’Keefe BR, Vojdani F, Buffa V, Shattock RJ, Montefiori DC, Bakke J, Mirsalis J, d’Andrea AL, Hume SD, Bratcher B, Saucedo CJ, McMahon JB, Pogue GP, Palmer KE. Proc Natl Acad Sci U S A. 2009; 14;106:6099-104.

To prevent sexually transmitted HIV, the most desirable active ingredients of microbicides are antiretrovirals that directly target viral entry and avert infection at mucosal surfaces. However, most promising antiretroviral entry inhibitors are biologicals, which are costly to manufacture and deliver to resource-poor areas where effective microbicides are urgently needed. Here, O’Keefe and colleagues report a manufacturing breakthrough for griffithsin, one of the most potent HIV entry inhibitors. This red algal protein was produced in multigram quantities after extraction from Nicotiana benthamiana plants transduced with a tobacco mosaic virus vector expressing griffithsin (GRFT). Plant-produced GRFT (GRFT-P) was shown as active against HIV at picomolar concentrations, directly virucidal via binding to HIV envelope glycoproteins, and capable of blocking cell-to-cell HIV transmission. GRFT-P has broad-spectrum activity against HIV clades A, B, and C, with utility as a microbicide component for HIV prevention in established epidemics in sub-Saharan Africa, South Asia, China, and the industrialized West. Cognizant of the imperative that microbicides not induce epithelial damage or inflammatory responses, the authors also show that GRFT-P is nonirritating and noninflammatory in human cervical explants and in vivo in the rabbit vaginal irritation model. Moreover, GRFT-P is potently active in preventing infection of cervical explants by HIV-1 and has no mitogenic activity on cultured human lymphocytes.

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Editors’ note: This study reports an exciting new development on the microbicide front - using Nicotiana benthamiana, a close relative of Nicotiana tobaccum (tobacco), and a tobacco mosaic virus vector to produce large quantities of a red algal protein, griffithsin, that is both directly virucidal to HIV and blocks cell-to-cell HIV transmission. This HIV entry inhibitor is unlikely to be absorbed systemically when applied topically and the vector used to produce it here is already manufacturing proteins used in clinical trials. Critically, griffithsin does not induce any of the proinflammatory cytokines known to recruit HIV target cells and promote HIV replication. These findings provide support in favour of griffithsin now advancing to human trials.

Disruption of Tight Junctions by Cellulose Sulfate Facilitates HIV Infection: Model of Microbicide Safety. Mesquita, P. Cheshenko N, Wilson S, Mhatre M, Guzman E, Fakioglu E, Keller M, and Herold B. J Infect Dis. 2009;200:599-608.

The lack of biomarkers that are predictive of safety is a critical gap in the development of microbicides. The present experiments were designed to evaluate the predictive value of in vitro models of microbicide safety. Changes in the epithelial barrier were evaluated by measuring transepithelial electrical resistance (TER) after exposure of human epithelial cells to candidate microbicides in a dual-chamber system. The significance of observed changes was addressed by challenging cultures with HIV and measuring the ability of virus to cross the epithelium and infect target T cells cultured in the lower chamber. Exposure to nonoxynol-9 (N-9) or cellulose sulfate (CS), but not 9-[2-(phosphonomethoxy)propyl] adenine (also referred to as tenofovir) or PRO2000, resulted in a rapid and sustained reduction in TER and a marked increase in HIV infection of T cells cultured in the lower chamber. Moreover, cellulose sulfate triggered nuclear factor kB activation in peripheral blood mononuclear cells and increased HIV replication in chronically infected U1 cells. Epithelial barrier disruption and enhanced viral replication may have contributed to the increased risk of HIV acquisition observed in phase 3 trials of nonoxynol-9 and cellulose sulfate. Expansion of in vitro safety testing to include these models would provide a more stringent preclinical assessment of microbicide safety and may prove to be more predictive of clinical outcomes.

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Editors’ note: The microbicide field had faced setbacks in clinical trials, including the unanticipated finding of increased HIV acquisition in one of two recent phase III clinical trials of cellulose sulphate. To date, preclinical safety studies have included in vitro (outside the human body) measurements of cell viability and effects on lactobacilli, rabbit vaginal irritation, and a few macaque studies, while phase I safety studies in humans have looked for signs of irritation, assessed colposcopic abnormalities, measured inflammatory cytokines, or cultured specific bacteria. This innovative study assessed changes in electrical resistance and effects on junctional proteins in the vaginal epithelium when each of four microbicides was applied. The authors found that polarized cells provide a relatively impervious barrier to HIV migration through the epithelium. It is encouraging that neither PMPA (tenofovir) nor PRO 2000 0.5% disrupted epithelial tight junctions or activated inflammatory pathways. Both of these candidates are currently in trials: CAPRISA 004 and MDP 301, respectively. The results of these trials will help better determine the place of this methodology in the safety evaluation of future microbicide candidates but based on this report it does warrant serious consideration.