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2Viral and Rickettsial Disease Laboratory, California Department of Public Health, ... 4Department of Chemistry, Massachusetts Institute of Technology, ...

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Reducing Infectivity of HIV Upon Exposure to Surfaces Coated With N,N-Dodecyl, Methyl-Polyethylenimine Stephen E. Gerrard,1,2,3 Alyssa M. Larson,4 Alexander M. Klibanov,4,5 Nigel K.H. Slater,1 Carl V. Hanson,2 Barbara F. Abrams,3 Mary K. Morris2 1

Department of Chemical Engineering and Biotechnology, BioScience Engineering Research Group, University of Cambridge, New Museums Site, Pembroke Street, Cambridge CB2 3RA, UK; telephone: þ44-1223-763969; fax: þ44-1223-334796; e-mail: [email protected] 2 Viral and Rickettsial Disease Laboratory, California Department of Public Health, Richmond, California 3 Division of Epidemiology, School of Public Health, University of California, Berkeley, California 4 Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 5 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts

ABSTRACT: The infectivity of high-titer, cell-free HIV in culture media and human milk is rapidly reduced upon exposure to polyethylene slides painted with the linear hydrophobic polycation N,N-dodecyl,methyl-polyethylenimine (DMPEI). Accompanying viral p24 protein and free viral RNA analysis of solutions exposed to DMPEI-coated surfaces suggests that virion attachment to the polycationic surface and its subsequent inactivation are the likely mechanism of this phenomenon. Biotechnol. Bioeng. 2013;110: 2058–2062. ß 2013 Wiley Periodicals, Inc. KEYWORDS: HIV; mother-to-child transmission (of HIV); breastfeeding; hydrophobic polycation; antiviral surface; polyethylenimine

Introduction Recently proposed virus processing technologies aim to reduce infectious viral titers in human milk, in particular those of HIV (Borkow et al., 2008, 2011; Gerrard et al., Correspondence to: S. E. Gerrard Contract grant sponsor: U.S. Army Research Office Contract grant number: W911NF-07-D-0004 Received 8 January 2013; Revision received 31 January 2013; Accepted 6 February 2013 Accepted manuscript online 21 February 2013; Article first published online 7 April 2013 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.24867/abstract) DOI 10.1002/bit.24867

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2012). Mother-to-child transmission (MTCT) of HIV in breastfeeding accounts for up to 40% (200,000) of infant infections of the disease, with 90% of these transmissions occurring in sub-Saharan Africa (Chasela et al., 2010). In the resource constrained regions where this form of HIV transmission occurs, there often is no safer option than for the mother to breastfeed and risk infecting her child (Kindra et al., 2012). One possible method to reduce MTCT in breastfeeding would be to pass infected milk through a device which reduces the infectious viral load while maintaining the nutritional and immunological benefits that the breast milk delivers to the infant. Such a device could be incorporated into a baby bottle or a nipple shield delivery system (NSDS) worn by the mother during feeding (Borkow et al., 2008, 2011; Gerrard et al., 2012). Conventional nipple shields are thin silicone devices placed over the breast which are used to aid the breastfeeding process for mother and child. They help premature children latch onto the nipple and can also be used to protect the nipple if it is sore (Riordan, 2005). The aforementioned NSDS could be used for anti-HIV applications where it contains an insert which could disinfect human milk. This could be accomplished by depositing an agent (an anti-viral or medication) into the fluid as the baby feeds. Alternatively, the NSDS could act on the fluid through a surface-immobilized disinfectant to reduce the infectious viral load in the breast milk that passes through, and subsequently decrease MTCT of HIV while avoiding the addition of foreign chemicals into the breast milk. ß 2013 Wiley Periodicals, Inc.

Herein we investigate the antimicrobial ‘‘paint’’ N,Ndodecyl,methyl-polyethylenimine (DMPEI; Klibanov, 2007) for its potential use as the antimicrobial component in an NSDS with the goal of reducing HIV viral load in human breast milk. This hydrophobic polycationic coating material has been demonstrated to avidly inactivate pathogenic bacteria and fungi, as well as to disinfect upon contact solutions containing influenza viruses, poliovirus, rotavirus, and herpes simplex viruses (Haldar et al., 2006; Hsu et al., 2011a; Klibanov, 2007; Larson et al., 2011, 2013). It has also been demonstrated to not leach from polyethylene and glass surfaces incubated in PBS and LB agar (Park et al., 2008; Haldar et al., 2006). Previously, we have shown through visualization by scanning electron microscopy of influenza viruses exposed to DMPEI-coated surfaces that virions adhere to the surface, followed by lysis during which a portion of viral RNA is released (Hsu et al., 2011b). To date, it is unknown whether DMPEI is active against HIV. In this study we analyze the effect of DMPEI on cell-free HIV, suspended in either culture media or human milk, to determine whether it too is susceptible to inactivation by this surface-immobilized hydrophobic polycation. Specifically, we carry out TZM-bl luciferase reporter infectivity assays, as well as viral capsid p24 protein and viral RNA assays, on washings of viral solutions that have come in contact with coated surfaces. This is to assess the reduction of infectious virus by DMPEI and the mode of inactivation. To test the viral sensitivity of HIV to DMPEI, 10 mL of high-titer, cell-free HIV (type 1 IIIB) that was suspended in either cell culture media or human milk was placed onto the center of a polyethylene slide coated with DMPEI. Another DMPEI-coated slide was then placed on top to sandwich the droplet, followed by placing a 0.1 lb weight on-top of the slide set (to ensure a good surface contact with the droplet). After a 15 min incubation at room temperature, the weight and top slide were removed, and 500 mL of cell culture media was pipetted over the exposed slide surface into a Petri dish. The collected wash was then pipetted over the slide 10 more times to ensure non-adhered viral particles would be washed off into the solution. This was then used again to wash viral particles off the other slide in the pair. As controls, uncoated polyethylene slides, as well as no slide, were used to determine the extent that polyethylene surface can reduce viral load of the solution. The wash was then collected from the Petri dish and assayed for infectivity via a TZM-bl infectivity assay. Additionally, washings were assayed for viral p24 protein and viral RNA to determine the fate of the viral particle after incubation with DMPEI coated surfaces. Note that although the HIV titers used in this work exceed those typically found in human milk (Hartmann et al., 2006), they allow a large range of viral reduction to be measured with the assays used. Control tests were also performed to ensure that no DMPEI leaching was occurring and resulting in antimicrobial behavior. Culture media not containing virus was

exposed to triplicate pairs of DMPEI coated and uncoated slides using the same procedure as for virus samples exposed to the slides. Wash samples from the slides were then exposed to HIV and measured for infectivity using TZM-bl cells. There was no statistical difference in reduction of infectivity between coated and uncoated slides (unpaired t-test, P > 0.05) and no cytotoxic effects were observed on TZM-bl cells compared to control samples for both slide types. The indistinguishable reduction in infectivity from coated and uncoated slides and the lack of TZM-bl cell damage suggests that no DMPEI leaching occurred. Additionally milk samples were similarly exposed to DMPEI coated and uncoated slides and no cytotoxic effects on TZM-bl cells compared to controls were observed. Analysis of the collected washings, in triplicate, using a TZM-bl infectivity assay demonstrated a statistically significant reduction of viral infectivity (unpaired t-test, one tailed) in cell culture media for coated slides (>3.3 log reduction, P < 0.0001) compared to a far smaller reduction for uncoated slides (0.47 log reduction P < 0.05; Fig. 1A). Controls using no slides indicated no statistical reduction in infectivity. The TZM-bl assay was also carried out with the washings of HIV suspended in human breast milk that were incubated between DMPEI coated slides (again, along with the appropriate controls, which demonstrated no statistically significant reduction in viral infectivity). The milk used had been previously determined to have low anti-viral behavior so as to not disrupt infectivity assays or act itself as a microbicidal agent against HIV. A statistically significant reduction of viral infectivity was also found in milksuspended virus for coated slides (>3.3 log reduction P < 0.0001) as compared to uncoated slides (0.42 log reduction P < 0.01; Fig. 1B). This stark reduction in viral infectivity from solutions incubated with DMPEI-coated slides as compared to uncoated, bare slides suggests a great loss of infectious HIV from solution when the virus encounters our hydrophobic-polycation coated surfaces. A 10-fold increase of viral titer in culture media was used in subsequent tests so that an absolute value of reduction in viral infectivity could be established. Tests were performed in triplicate and analyzed again with the TZM-bl infectivity assay. For this higher titer, a significant average reduction of viral infectivity was found on coated slides (1.0 log reduction, P < 0.0001) whereas no statistically significant reduction was observed for uncoated slides (0.06 log reduction, P > 0.05; Fig. 2). For this higher titer of HIV, viral RNA and viral p24 protein were also measured from the slide wash to better understand the fate of the viral particle after contact with DMPEI-painted surfaces. Incubation with coated slides produced statistically significant reductions in all measurements (i.e., p24, RNA, and TZM-bl infectivity) and there was no difference between the reductions in viral p24 protein (0.52 log, P < 0.01) and viral RNA (0.51 log, P < 0.001; paired t-test, two tailed, P > 0.05; Fig. 2). However, there was a significant difference for both p24

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Figure 1. The reduction of viral infectivity of cell-free HIVIIIB (8.0  109 RNA copies/mL) suspended in (A) culture media or (B) human milk after a 15 min contact between two polyethylene slides either coated with N,N-dodecyl,methyl-polyethylenimine (DMPEI) or a plain uncoated polyethylene slide. Results are depicted as the average of three repeat experiments using separate slides for both coated and uncoated surfaces. A statistically higher reduction in viral infectivity for coated slides compared to uncoated ones was found for HIV suspended in both culture media and human milk (unpaired t-test, P < 0.05). For the coated slide in both viral suspensions, the reduction of infectivity exceeded the lower limit of detection of the TZM-bl assay which correlated to a >3.3 log reduction. The standard error for repeat experiments is shown.

and viral RNA assays when compared to the reduction in viral infectivity from the TZM-bl assay (1.1 log, P < 0.0001; TZM-bl vs. p24 reduction P < 0.01, TZM-bl vs. RNA reduction P < 0.05, paired t-test two-tailed). Smaller reductions between the measurements on uncoated slides were found, which were statistically insignificant from each other (paired t-test, two tailed, P > 0.05; Fig. 2). These statistically significant reductions in infectious viral titer, p24 protein, and RNA for DMPEI coated slides as compared

Figure 2. The reduction of viral infectivity (8.0  1010 RNA copies/mL), viral p24 protein, and viral RNA of cell-free HIVIIIB suspended in culture media after a 15 min contact between two polyethylene slides, either coated with DMPEI (gray bars) or a plain uncoated surface (white bars). A statistically higher reduction of viral infectivity was found on coated slides compared to both viral p24 protein and RNA values (paired t-test, two tailed P < 0.05). There was no statistically significant difference between all assays for uncoated slides or between p24 protein and RNA for coated slides (paired t-test, two tailed P > 0.05). Results are displayed as the average of three repeat experiments using separate slides for both coated and uncoated surfaces, with the standard error shown for repeat experiments.

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to uncoated slides demonstrates the disinfecting ability of DMPEI towards HIV either suspended in media or human breast milk. This study demonstrates that when HIV at high titer in either human milk or culture media is exposed to DMPEIcoated surfaces it rapidly loses its infectivity. It is important to note that there will be residual levels of p24 protein and viral RNA within the viral solution prior to exposure to DMPEI, as well as a large proportion (approximately 3,000:1) of non-infectious to infectious viruses, thus conclusions on the specific mechanism of anti-viral behavior of immobilized DMPEI on HIV can only be hypothesized at this point. The higher reduction in infectivity as compared to a smaller reduction in viral RNA and p24 protein suggests the possibility of irreversible virion attachment to the coated slide. This, accompanied with partial viral lysis of the lipid membrane, could result in the release of the p24 protein capsid and RNA into the viral wash, leading to higher detectable amounts of p24 and RNA than infectivity loss predicts. This would be consistent with our previous observations of similar behavior occurring with influenza virion interactions with immobilized DMPEI (Hsu et al., 2011b). In a clinical setting a DMPEI coated fiber membrane used in a NSDS is likely to have a reduced capacity for viral inactivation over the duration of an infant feed, if virus is binding to the membrane surface. Sufficient loading of DMPEI to the fibers would be needed to ensure a high enough capacity was available for extensive viral inactivation throughout the feed. This work suggests that the virucidal behavior of DMPEI against phospholipid membrane viruses is not specific to influenza and herpes simplex viruses (Hsu et al., 2011b;

Larson et al., 2013), but also holds for HIV. It also demonstrates that when high titer HIV is suspended in human milk, a rapid reduction in viral infectivity is observed, indicating that milk components might not significantly interfere with DMPEI’s virucidal properties. Consequently, a DMPEI coated membrane may be suitable for disinfecting HIV-containing human milk with a NSDSlike flow-through device. Additionally, its anti-viral behavior may find applications in environments where there is the need for a broadly acting, non-specific anti-viral material.

Materials and Methods Chemicals DMPEI was synthesized from linear 217-kDa polyethylenimine as previously described by Hsu et al. (2011a). All chemicals and reagents for synthesis were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO) and used without further purification. Polyethylene sheets were from McMaster-Carr (Elmhurst, IL). Cells and Viruses Cell-free HIV-1 type IIIB was concentrated from infected H9/HIVIIIB cells expressing the virus (provided by the NIH AIDS Research and Reference Reagent Program Cat. No. 400). TZM-bl cells (also provided by the NIH AIDS Research and Reference Reagent Program Cat. No. 8129) were incubated at 36.58C and 5% CO2 in a Sanyo incubator for assaying sample infectivity (i.e., when exposed to HIV) and also when being cultured. Cells and virus were grown and suspended in a culture media primarily composed of Dulbecco’s Modified Eagle media (DMEM) with high glucose and 15% fetal bovine serum (Invitrogen, Carlsbad, CA). Human Milk Mature human milk was obtained from a healthy anonymous HIV-negative donor over the age of 18 from the Milk Bank, Santa Clara Valley Medical Centre (San Jose, CA). The donor sample was selected from 13 donors and had no detectable antimicrobial or cytotoxic behavior, so any anti-viral action upon HIV samples could be primarily attributed to DMPEI. Slide Preparation Polyethylene slides were painted with solutions of DMPEI as described in Haldar et al. (2007) and Larson et al. (2011). Briefly, polyethylene sheets were cut into 2.5  2.5 cm2 and sonicated in 70% ethanol. Slides were allowed to dry and one side was painted, in triplicate, with a 50 mg/mL solution of DMPEI in chloroform. Solvent was allowed to evaporate between successive paintings.

TZM-bl Infectivity Assay Triplicate 25 mL samples and serial dilutions of 1 to 5, 10, 50 (vol.) of the collected viral wash exposed to coated and uncoated slides were separately added to flat bottom 96-well plates, followed by 25 mL of cell culture media and 50 mL of TZM-bl cells suspended in cell culture media at 2  105 cells/ mL. Just prior to sample additional DEAE Dextran was added to the TZM-bl cells to a final concentration of 30 mg/ mL and samples were incubated for 3 days. A D-Luciferin potassium salt was then added to all samples, 100 mL of each sample transferred to a black flat-bottom plate and luminescence was measured using a Glomax1 96 Microplate Luminometer (ProMega, Madison, WI). Measurements were then correlated against starting virus content not exposed to slides (eight serial dilutions of 1–2.5 and 1–10) to compare loss of viral infectivity for slides. P24 Capsid Protein ELISA Viral p24 protein concentrations were measured using an in-house enzyme immunoassay. Using this method, detectable ranges of p24 protein can be measured from 20 to 10,000 pg/mL. Triplicate samples of the viral wash and three serial dilutions were assayed for p24 content and compared against the original values of non-exposed starting material. Viral RNA Assay Viral RNA was measured in triplicate for each test using COBAS1 AmpliPrep/COBAS1 TaqMan1 HIV-1 Test, v2.0 (Roche, Pleasanton, CA). After preliminary tests to determine the approximate range of RNA reduction, samples were diluted to 6 and 7 logs of their original concentration and compared against the non-exposed viral solution’s RNA content. We are grateful to the Bill and Melinda Gates Foundation (Grand Challenges Exploration Initiative), the Clinton Foundation (Clinton Global Initiative University), the U.K. EPSRC, Cambridge University and King’s College (University of Cambridge), Pembroke College (University of Cambridge)—UC Berkeley Exchange, and the International Development Design Summit for financial support and advice. In addition, this research was partially supported by the U.S. Army Research Office under contract W911NF-07-D0004. We thank David McNally and Krishnaa Mahbubani of the Bioscience Engineering Group, Department of Chemical Engineering and Biotechnology, University of Cambridge, UK as well as Peter Patiris, Leo Oceguera and Haynes Sheppard of the California Department of Public Health, Richmond for advice. We also thank Pauline Sakamoto of the Milk Bank, Santa Clara Valley Medical Centre (San Jose, CA) for coordinating use of human milk samples. Stephen Gerrard is an inventor of the nipple shield delivery system (US patent application no. 12/536,219 and patent pending PCT/US10/44589, see http://justmilk.org). Alyssa M. Larson is a recipient of the Martin Predoctoral Fellowship.

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