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Chemokine Analogues Show Suitable Stability for Development as Microbicides

Cerini, Fabrice*; Landay, Alan PhD; Gichinga, Carolyne; Lederman, Michael M MD; Flyckt, Rebecca MD§ ; Starks, David MD§ ; Offord, Robin E PhD; Le Gal, François PhD; Hartley, Oliver PhD*

JAIDS Journal of Acquired Immune Deficiency Syndromes: December 2008 - Volume 49 - Issue 5 - p 472-476
doi: 10.1097/QAI.0b013e31818c953f
Basic Science
Free
SDC

New prevention strategies are urgently needed to slow the spread of the HIV/AIDS pandemic, and in the absence of an effective vaccine, there is hope that “microbicides”-HIV inhibitors applied to mucosal surfaces before sexual intercourse-may be able to make an impact.

Because developing countries are at the center of the epidemic, affordability and stability during storage are key criteria for candidate microbicides. Furthermore, because formulation strategies that provide long-duration protection after a single dose may enhance acceptability and compliance, stability in the vaginal environment and in the presence of semen should also be considered.

PSC-RANTES, a human chemokine analog, has shown promise as a candidate microbicide, but because it contains nonnatural structures that necessitate chemical synthesis steps, it is not suitable for production at a feasible cost and scale for general distribution in developing countries. We have recently developed 2 new fully recombinant chemokine analogs, 5P12-RANTES and 6P4-RANTES, which show equivalent anti-HIV activity to PSC-RANTES. In this study, we tested the stability of these molecules under conditions related to use as microbicides. Our results suggest that stability issues will not present a major obstacle to the further development of these promising molecules as microbicides.

From the *Department of Structural Biology and Bioinformatics, Faculty of Medicine, University of Geneva, 1 rue Michel Servet, Geneva, Switzerland; †Department of Immunology/Microbiology, Rush University Medical Center, Chicago, IL; ‡Department of Medicine; and §Department of Obstetrics and Gynecology, Case Western Reserve University, University Hospitals-Case Medical Center, 2061 Cornell Road, Cleveland, OH; Mintaka Foundation for Medical Research, 14 chemin des Aulx, Plan-les-Ouates, Geneva, Switzerland; and ¶Department of Obstetrics and Gynecology, Geneva University Hospital, 30 Boulevard. de la Cluse, Geneva, Switzerland.

Received for publication July 29, 2008; accepted September 3, 2008.

Supported by grants from the US National Institutes of Health (PO1 AI 51649-01 and R21AI071935), the Swiss National Science Foundation, the Mintaka Foundation for Medical Research (Switzerland), the Esperanza Medicines Foundation (Switzerland), and the La Jolla Foundation for Microbicide Research.

Correspondence to: Department of Structural Biology and Bioinformatics, Faculty of Medicine, University of Geneva, 1 rue Michel Servet, 1211 Geneva 4, Switzerland (e-mail: oliver.hartley@medecine.unige.ch).

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INTRODUCTION

According to the most recent estimates, the worldwide HIV epidemic currently affects 33 million people, with 2.5 million new infections per year.1 Prevention methods that could slow the spread of the epidemic are urgently needed, but recent results have led to the suggestion that many more years will be required before an effective HIV vaccine is produced.2 There is hope that strategies of topical prevention involving the use of substances known as microbicides3,4 might have an earlier impact, but recent failures in large scale microbicide trials5,6 have highlighted the need to accelerate the clinical development of promising new candidates.

Previous microbicide candidates are thought to have failed owing to problems with safety and lack of efficacy,5-7 but in addition to satisfying these key requirements, new microbicide candidates will need to be appropriate for use in the poorest countries of the world.3,4,8 Hence, they must be both inexpensive to produce and stable to storage in the absence of an effective cold chain. Furthermore, because success is likely to depend on the delivery of long-duration protection after a single dose,3,4,8 new microbicide candidates will need to show acceptable stability in the vaginal environment and in the presence of semen.

Certain human chemokine proteins exhibit intrinsic anti-HIV activity9 through their ability to block the HIV coreceptor CCR5, and a number of N-terminally modified analogues of these proteins with very much higher antiviral potency have been developed.10-16 PSC-RANTES is the most potent of the anti-HIV chemokine analogs identified so far14 and is remarkable in that it is capable, alone, of providing full protection in the macaque vaginal challenge model that is routinely used to evaluate candidate microbicides.17 However, because PSC-RANTES contains nonnatural structures that would necessitate chemical synthesis steps, it has been argued that its production costs would be prohibitively expensive.18 To address this problem, we have reengineered 2 new fully recombinant proteins from PSC-RANTES, called 5P12-RANTES and 6P4-RANTES. Both proteins show equivalent potency to PSC-RANTES in viral replication assays using physiological target cells and relevant R5-tropic viral strains. Like PSC-RANTES, 6P4-RANTES is a strong CCR5 agonist that acts by inducing intracellular sequestration of CCR5. In contrast, 5P12-RANTES does neither detectably activate G protein-linked signaling via CCR5 nor induce intracellular sequestration of the receptor [H. Gaertner, F. Cerini, G. Kuenzi, et al, Highly potent, fully recombinant anti-HIV chemokines: re-engineering a low-cost microbicide. Proc Natl Acad Sci, 2008; In press]. Furthermore, when tested in a standard macaque vaginal challenge model, both 5P12-RANTES and 6P4-RANTES provided full protection (each compound protected 5 of 5 animals) (R. Veazey, B. Ling, L. Green, et al, unpublished data, 2008). Most importantly, because they are comprised exclusively of natural, coded amino acids, they also have the advantage of being accessible to the ultralow-cost, high-volume biosynthetic manufacturing techniques19 that are currently being used to produce GMP products at a cost and scale that would be appropriate for worldwide distribution of protein-based microbicides.

In this study, we performed a preliminary evaluation of the stability of 5P12-RANTES and 6P4-RANTES under conditions related to their use as microbicides: storage at elevated temperature, exposure to vaginal pH, incubation with human cervicovaginal lavage (CVL) fluid, and incubation with human semen. In each case, stability was assessed using a highly sensitive biological assay based on the ability of the chemokines to prevent the HIV entry process. For the experiments using human CVL and semen samples, the stability of PSC-RANTES was also determined for comparison.

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METHODS

Cell Fusion Assay

This assay was carried out as described in Hartley et al14 using the HeLa-P5L10 and HeLa-Env-ADA20 cell lines. HeLa-P5L cells were seeded in 96-well plates (104 cells per well). Twenty-four hours later, medium was removed and replaced with medium containing 104 HeLa-Env-ADA cells per well plus chemokines. Chemokines were used at either 9 concentrations (5-fold serial dilutions from a highest concentration of 500 nM), except in the case of the human CVL samples, where 5 concentrations were used (10-fold serial dilutions from a highest concentration of 100 nM). After a further 24 hours, cells were washed once in phosphate-buffered saline, lysed, and assayed for β-galactosidase activity by the addition of the colorigenic substrate chlorophenol red-β-D-galactopyranoside. Results were expressed as 100 × [mean absorbance (treated) − mean absorbance (no envelope cells)]/[mean absorbance (no chemokine) − mean absorbance (no envelope cells)]. Measurements were performed in triplicate for each independent experiment, and pIC50 values [−log(IC50 value expressed in mol/L)] obtained from dose-inhibition curves were fitted using Prism software (GraphPad).

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Human CVL Samples

Healthy women attending Gynecology clinic for routine evaluation were asked to participate in the study. After signing informed consent, and with the patient in the dorsal recumbent position, 20 mL of sterile saline was introduced into the vaginal vault with a plastic syringe and then immediately aspirated. Samples were tested either immediately or after centrifugation to remove particulate matter. To 200 μL of CVL fluid was added 50 μL of chemokine analogue in citrate buffer pH 5.5 for a final analog concentration of 10 μM. After incubation for 24 hours in a 37°C water bath, the samples were frozen at −80°C before assay.

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Human Semen Samples

This work was performed according to a protocol approved by the Geneva University Hospital Ethics Committee. Ten men (33.4 ± 4 years old) who were routinely examined before a medically assisted procreation trial gave their consent to participate in this study. The semen parameters for the participants (mean volume 3.82 ± 0.89 mL, mean spermatozoa concentration 6.5 ± 3.5 × 107 spermatozoa/mL, and mean progressive motility 55.5% ± 7.8%) were within the normal range.21,22

After a 30- to 45-minute incubation in a 37°C water bath (for postliquefaction samples), duplicate aliquots of 200 μL each were transferred into a 1.5-mL microtube containing 50 μL of 5P12-RANTES or 6P4-RANTES and incubated for 24 hours in a 37°C water bath. Samples were then frozen at −80°C before subsequent analysis.

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RESULTS

Stability of Biological Activity to Storage at Elevated Temperature

To study the temperature stability of the proteins, standard laboratory stock solutions of 5P12-RANTES and 6P4-RANTES (100 μM in pure water: the intrinsic pH of such solutions is approximately pH 4.8) were either stored frozen or incubated in a water bath for (1) 24 hours at 55°C or (2) 7 days at 40°C. Biological activity (pIC50) was then determined using the cell fusion assay (Fig. 1). No detectable change in activity for either compound under either condition was apparent even when no bacteriostat was present. PSC-RANTES shows comparable stability in experiments performed under similar conditions (O. H. REO, et al, unpublished results).

FIGURE 1

FIGURE 1

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Stability to Exposure to Vaginal pH

The pH of vaginal secretions varies between pH 3.8 and 4.5.23 We chose to test the stability of the chemokine analogues at pH 4.0. Stock solutions of 5P12-RANTES and 6P4-RANTES (100 μM in pure water) were either stored frozen or diluted 1:10 in 50 mM of citrate buffer, pH 4.0 and left at ambient temperature for 24 hours. Biological activity (pIC50) was then determined using the cell fusion assay (Fig. 2). No detectable changes in activity were apparent.

FIGURE 2

FIGURE 2

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Stability to Incubation With Human CVL Samples

To assess the potential for degradation of chemokine analogues by enzymes present in the vaginal fluid, we chose test conditions that would favor detection of enzyme activity (relatively low substrate concentration, long incubation time, solutions buffered to low pH). Hence samples of chemokine analogues (10 μM made up in 50 mM of citrate buffer, pH 5.5) were incubated with CVL samples for 24 hours at 37°C before evaluation in the cell fusion assay.

In a first experiment, untreated samples were tested alongside centrifuged samples obtained from the same 3 donors. Control samples were incubated under the same conditions with saline vehicle alone (Fig. 3). Incubation of 5P12-RANTES and 6P4-RANTES with both whole and centrifuged CVL from all 3 donors had no detectable effect on biological activity even without the presence of a bacteriostat. In contrast, incubation with one of the whole CVL samples (donor 507) led to a modest (approximately 3-fold) reduction in the biological activity of PSC-RANTES.

FIGURE 3

FIGURE 3

In a second series of experiments, stability of 5P12-RANTES and 6P4-RANTES was tested using whole CVL samples from a second, larger group (n = 20) of healthy donors (Fig. 4). In these experiments, CVL samples capable of causing detectable degradation were identified. The CVL sample from donor P reduced the biological activity of both 5P12-RANTES and 6P4-RANTES by over 30-fold under the conditions used, and samples from donors I, Q, and R reproducibly reduced the biological activity of both chemokine analogues, albeit to a lesser extent (approximately 3-fold). None of the other samples caused detectable degradation of either chemokine analogue. Hence from a total of 23 CVL samples from healthy donors (3 from the first experiment and 20 from the second), 3 caused detectable degradation under the conditions used.

FIGURE 4

FIGURE 4

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Stability to Incubation With Human Semen Samples

We next set up a series of experiments to determine the stability of the chemokine analogues in the presence of human semen. Because a protease-mediated liquefaction process takes place in semen 15-60 minutes after ejaculation,24,25 we began with a pilot experiment to ascertain if the capacity of freshly obtained semen samples to degrade the chemokine analogues is modified during the liquefaction process. Using samples from 3 donors, we performed parallel incubations before and after liquefaction. No differences were apparent (data not shown), so we opted to perform subsequent work using postliquefaction samples. Data were collected on samples from 10 different donors. Under the conditions tested, none of the 10 samples showed any detectable degradation of activity of 6P4-RANTES, and only 1 sample (donor no. 5) resulted in a detectable reduction in activity of 5P12-RANTES (approximately 30-fold). In contrast, most samples degraded the activity of PSC-RANTES at levels ranging from approximately 4-fold (eg, sample no. 9) up to approximately 10-fold (eg, sample no. 2).

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DISCUSSION

In addition to exhibiting strong anti-HIV efficacy and an excellent safety profile, agents to be used as microbicides must meet the following criteria: (1) production at a cost and scale appropriate for worldwide use, (2) sufficient stability upon storage in the absence of a cold chain, and (3) sufficient stability in the vaginal environment and in the presence of semen.3,4,8 Both in vitro and in vivo evaluation of 2 new chemokine analogues, 5P12-RANTES and 6P4-RANTES, demonstrate that they are among the most effective anti-HIV agents currently under evaluation as microbicides. Preliminary studies (F. Cerini, O. Harley, R. E. Offord, unpublished data) suggest that, as expected, these fully recombinant proteins are compatible with biosynthetic production approaches used to produce proteins at an industrial scale at ultralow cost.19

In this study, we set out to ascertain if any major stability issues, either during storage or use, could be identified that would be likely to affect the further development of these molecules as microbicides. We chose to use a sensitive biological assay directly related to a key function (inhibition of HIV infection) as read out. The activity of both molecules does not seem to be particularly sensitive to elevated temperature (Fig. 1) or to prolonged exposure to vaginal pH (Fig. 2). Similarly, the activity of both molecules showed excellent stability after incubation with human CVL (Figs. 3 and 4) and with human semen (Fig. 5).

FIGURE 5

FIGURE 5

Because we used a biological assay to assess stability, we cannot exclude the possibility that modifications to the proteins that do not affect anti-HIV activity occurred. Use of appropriate analytical techniques for the detection and characterization of such products will be a necessary component of the further preclinical development of these molecules. The preliminary studies of stability in the presence of human CVL and semen were carried out on samples obtained from healthy volunteers living in North America and Europe. It will be important at a later stage to determine whether similar high levels of stability will be obtained when samples are incubated with CVL samples from people living in countries at the center of the HIV epidemic and in samples from women suffering from vaginal conditions (sexually transmitted diseases and bacterial vaginosis) that may enhance the risk of HIV acquisition.

Overall, this preliminary study failed to detect any major stability problems of a kind likely to provide an insurmountable obstacle to the further development of these promising molecules as microbicides.

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Keywords:

CCR5; chemokine; HIV/AIDS; microbicide; prevention; stability

© 2008 Lippincott Williams & Wilkins, Inc.