Antiviral drug development is fundamentally more challenging than antibiotic development (e.g. against bacteria), due to rapid viral biochemical mutation that negates drug efficacy. It appears that directly attacking a virus will always be a struggle.
Consequently, it makes sense to think of fundamentally different approaches to antiviral treatment. Instead of directly attacking a virus, why not target a cellular biochemical system essential to viral infection and propagation?
Nicole Zitzmann (University of Oxford, United Kingdom) and coworkers have taken this approach to target hepatitis B, hepatitis C, and HIV infections. Their liposomes hindered viral propagation in infected cells, as well as viral infection of uninfected cells, by lowering cellular cholesterol levels.
Why target cholesterol biosynthesis?
Why would reducing cholesterol biosynthesis have any impact on a virus? Some viruses critically depend upon cholesterol and lipids for survival.
For example, HIV and hepatitis B and C utilize them for cellular entry, and hepatitis C and HIV require them for assembly within the cell. Despite this promise, clinical efforts at combating viral infections by inhibiting cholesterol have essentially failed, possibly due to cellular compensation for the cholesterol inhibition.
Future efforts may be more successful if cholesterol biosynthesis is targeted more effectively. This was the goal of Zitzmann and coworkers.
The indirect antiviral agent.
The scientists' weapon of war is the humble liposome, in this case microscopic spherical assemblies comprised of polyunsaturated lipids (multiple double bonds in the lipid chains). The liposomes can enter cells, and are targeted to the endoplasmic reticulum (a cellular subcompartment, important in protein synthesis and many other functions).
It's important to note that the liposomes do not contain drugs within them, in this research. Future research may utilize drugs in combination with the liposomes for enhanced antiviral activity.
Targeting cholesterol biosynthesis.
The scientists found that a liposome concentration of 50 micromolar (measured by the lipid content) was the highest utilizable concentration that was still essentially nontoxic to their cells. All further studies were carried out at this concentration.
Tested against lab-cultured liver and blood cells, cholesterol was reduced by somewhere between 33% and 54%, depending on the type of cholesterol and the specific type of cell. This demonstrates significant cholesterol inhibition regardless of the cell type.
It's possible that cholesterol inhibition was effected by activation of an enzyme (sphingomyelinase) critical to cholesterol removal from the cell membrane, in response to an influx of polyunsaturated lipids (i.e. the liposomes). The liposomes were superior to lovastatin (a drug used to control cholesterol levels), at nontoxic concentrations, in terms of cholesterol inhibition, which didn't significantly affect sphingomyelinase activity.
The scientists tweaked their liposomes with a synthetic polymer, poly(ethylene glycol), which is commonly used to improve liposome biocompatibility. Similar results were obtained, i.e. poly(ethylene glycol) is not needed.
Liposomes targeted to the general cell interior, as opposed to the endoplasmic reticulum, weren't effective. However, this isn't evidence that the endoplasmic reticulum is the only effective target for inhibiting intracellular cholesterol production.
Now, on to the highlight of this research. The scientists' liposomes dramatically hindered viral activity.
Liposomes inhibited viral secretion by somewhere between 22% and 41%, depending on the virus and cell type. Viral infectivity was reduced by somewhere between 50% and 91%.
In contrast, treatment with lovastatin did not affect hepatitis C secretion from liver cells, and only reduced hepatitis infectivity by 48%. Clearly, the scientists' liposomes are more effective than levostatin at inhibiting the viruses, as expected due to lovastatins' inferior cholesterol targeting capacity.
Viral infectious levels were returned to normal by adding cholesterol to the cells. Viral spread is clearly closely related to intracellular cholesterol levels.
Pretreating noninfected cells with the liposomes reduced hepatitis C viral entry into cells by 94%, and that of HIV by 64% (the experiment could not be performed in the cells used for hepatitis B infection). Thus, not only do the liposomes hinder the spread of viruses from infected cells, they also hinder viral infection of healthy cells.
Further experiments demonstrated that hepatitis C infection was hindered by both reduced cholesterol in the cell membranes, as well as reduced expression (on the cell surface) of the protein CD81, important for viral attachment to the cell. No proteins important for HIV attachment to the cell were found to be altered by liposome treatment, but lipid rafts were less prevalent in the cell membrane.
Zitzmann and coworkers have developed a novel and extraordinarily useful method of indirectly targeting viral infections, namely by inhibiting cholesterol biosynthesis. This approach is unlikely to be thwarted by viral adaptation, due to critical viral dependence upon cholesterol for cellular infection.
Furthermore, this approach can be readily adapted to include drugs within the liposomes, especially for targeting simultaneous viral infections which are less dependent upon intracellular cholesterol. Note that this advance is limited to viral infections which need cholesterol to infect and spread to other cells.
Nevertheless, HIV and hepatitis infections are a huge health burden, and taking them out is a worthy goal in itself. This research is complimentary to that aimed at cheaply synthesizing topical anti-HIV agents; perhaps such drugs can be used in combination with the liposomes described herein to dramatically improve the efficacy of standard HIV treatment.
NOTE: The scientists' research was funded by the Oxford Glycobiology Endowment, the Canadian Institutes of Health Research, United Therapeutics Corporation, and the Consiliului National al Cercetarii Stiintifice din Invatamantul Superior.
for more information:
Pollock, S.;, Nichita, N. B.;, Böhmera, A.;, Radulescu. C.;, Dwek, R. A.;, & Zitzmann, N. (2010). Polyunsaturated liposomes are antiviral against hepatitis B and C viruses and HIV by decreasing cholesterol levels in infected cells Proceedings of the National Academy of Sciences, 107 : 10.1073/pnas.1009445107