Important Considerations in Antiviral Testing
The COVID-19 pandemic kickstarted a renewed interest in antiviral development within the pharmaceutical and personal care industries. But even before the COVID-19 pandemic viral infections have been a major public health burden, with children in developed countries alone experiencing an average of 6-10 cases of viral respiratory infections per year [1].
Viral infections begin when virus particles bind to and enter our cells. Once inside, the virus uses the cell to make more copies of itself before killing its host and in the process, allow the newly made virus particle to be released, to go on to infect more cells. The presence of viral components and dead cells elicits a strong response from the body’s immune system, which helps to clear the infection. However, the immune response can sometimes spiral out of control, leading to serious complications such as organ failure and even death. For these reasons, stopping the virus early on is an important pharmaceutical strategy.
One of the most common antiviral strategies is to directly inactivate the virus, rendering it unable to infect cells. Some of the pharmaceutical agents that employ this strategy can be highly specific for a given virus species, such as an antibody that binds to a specific surface protein on the viral particle, or a small molecule that disrupts a key enzyme in the virus reproductive cycle. Other antivirals have broad, non-specific activity, such as oxidation or disruption of viral structure, that allow them to be highly effective across a range of different types of viruses.
No matter the mode of action, the first step in the development of a new antiviral test article is to perform in-vitro testing to determine its optimal antiviral concentration [2]. The workflow for such a test is shown schematically in the figure below and begins with exposing the virus to different concentrations of the test article for a set amount of time (steps 1 and 2). After the exposure, a neutralizer is added to halt antiviral activity (step 3). The mixture of virus, test article, and neutralizer is then added to pre-prepared host cells to allow any remaining viable viral particles to infect the cells (step 4). Once the infection incubation is complete, the mixture is removed and fresh cell culture media is added to the cells (step 5). The antiviral effectiveness of the test article at different concentrations is determined by comparing host cell viability in the test article + virus group to the virus only control group using a viability dye such as MTS (step 6).
While the test appears simple, there are a number of factors to consider during its setup and execution to ensure the results represent true viral inactivation, and that false negative and positive results are accounted for with the appropriate controls. These controls include:
Virus infection control. This control provides the baseline for cell death in the absence of test article treatment. The virus stock should be tested prior to its use in the assay to ensure infectivity. At Emery Pharma all virus stocks are tested before use in an assay to ensure a consistent baseline in virucidal assays.
Neutralization control. The use of a neutralizer to inactivate an antiviral may seem counterintuitive, but it is an important control to include to ensure the validity of the test results. If a test article continues to exert antiviral activity beyond the desired contact time against the virus, it can produce a false positive result. By ensuring the test article has been fully inactivated by the neutralizer, the infectivity of any surviving virus can then be accurately assessed.
To set up the neutralization control, the test article is combined with the neutralizer solution. Virus is then added to the mixture. If the test article has been fully neutralized the virus should exhibit the same level of infectivity as the virus infection control.
The selection of a neutralizer is dependent on the chemical nature and mode of action of the test article to be tested. Emery Pharma’s biology team works closely with our chemistry team to help our clients identify the most appropriate neutralizer for their test article.
Cytotoxicity control. Cytotoxicity control is crucial in antiviral assays as it ensures the test article and neutralizer do not negatively affect the viability of the host cells and lead to false negative results. In this control group, the test article and neutralizer are applied to the host cell in the absence of a virus. The viability of treated cells is then compared to untreated control cells using MTS.
Cytotoxicity assessment should ideally be performed prior to antiviral assay to determine the appropriate nontoxic concentrations for both test articles and neutralizer. However, this is not always possible due to timeline and other constraints. In such instances, cytotoxicity controls can be run concurrently with the antiviral assessment.
Antiviral testing sits at the nexus of cell biology, virology, and chemistry, which are areas that the Emery Pharma team have extensive expertise and experience in. No matter the novelty or complexity of your antiviral agent, our team can help you obtain meaningful insight into your product’s antiviral capabilities. Contact us today to find out how we can help you drive your innovation to the next level.
References
- Pavia, AT. Viral Infections of the Lower Respiratory Tract: Old Viruses, New Viruses, and the Role of Diagnosis. Clin Infect Dis. 2011 May 1;52(Suppl 4):S284–S289. doi: 10.1093/cid/cir043
- Jekle A, Rani SA, Celeri C, Zuck M, Xu P, Wang L, Najafi-Tagol K, Anderson M, Stroman D, Debabov D. Broad-spectrum virucidal activity of (NVC-422) N,N-dichloro-2,2-dimethyltaurine against viral ocular pathogens in vitro. Invest Ophthalmol Vis Sci. 2013 Feb 19;54(2):1244-51. DOI: 10.1167/iovs.12-10700
