In this three-blog series, we present the typical workflow of an antiviral discovery program, from the initial identification of antiviral molecules in vitro (Part 1) to preclinical testing (Part 2) and assessing the onset of drug resistance (Part 3). These early stages of drug discovery are critical to bringing any compound to the clinic. Given the relatively lower costs of the R&D stages when compared to clinical studies, it is important that every step of the process is optimized to select only the most promising compounds. In this third and final blog, we discuss the preclinical assessment of virus resistance.
When the virus fights back
Rapid and high-copy viral replication and low fidelity of the viral replication machinery, as well as several host factors, can complicate antiviral discovery efforts. These traits in combination allow a virus to rapidly test different combinations of mutations until a strategy for escaping the antiviral barrier is discovered. Viruses that evolve to escape a drug or a vaccine are called resistant mutants. How many mutations are needed to develop resistance? This depends on a variety of factors, including the virus, the treatment, and the target. A virus with a low threshold for resistance development will only require one or two mutations to escape a treatment, while a virus with a high threshold will require multiple mutations to select for resistance. The second scenario is, of course, more desirable and this is thought to be the case for host-targeted antivirals: evolving a new way to adapt to a cell where a specific protein is blocked or an entire pathway is inhibited requires a high mutation rate that is inevitably accompanied by high evolutionary costs and decreased fitness. Most drugs, however, target viral components; for these the number of mutations required to develop resistance is generally much lower and represents one of the biggest problems in antiviral discovery.
In vitro assessment of resistant mutants
While resistant mutants developed in vitro might not be entirely representative of the population(s) selected in vivo or during clinical trials, assessment of virus resistance is a requirement to progress an antiviral to Phase I, and it can give important insights into the genetic threshold for resistance development (discussed above). Two methods are generally used to encourage the development of resistant mutants. In the first, a high virus inoculum is passaged several times in the presence of a fixed concentration of a drug. Multiple cultures can be used in parallel to test different concentrations. Alternatively, a low virus inoculum is passaged several times in the presence of different concentrations of products, starting from concentrations close to the IC50. The choice of assay will depend on the virus replication rate and the number of genome copies produced in tissue culture conditions. As these tend to be lower than in vivo, serial passaging is generally required. Whichever assay is chosen, it should be repeated for different strains (ideally including primary/clinical isolates), in different cell lines (ideally including relevant primary cells), and with different drug concentrations.
Genotypic and phenotypic analysis
Once resistant mutants have been isolated, the next step is to identify the mutations (genotypic analysis) and determine their contribution to resistance (phenotypic analysis). Genotypic analysis, besides revealing virus mutants, can also help to confirm the compound target, as mutations able to confer resistance will accumulate where the drug acts. Most commonly, Sanger sequencing is used to determine the mutation rate across the entire genome for small viruses, or in the proximity of the target area for larger viruses. The main limitation of this method is its sensitivity, as rare mutants representing less than 20% of the population typically cannot be identified. More recently, Next Generation Sequencing has introduced the power of single-molecule amplification and sequencing to the field of antiviral development, allowing the identification of resistant mutants that represent less than 1% of the isolated population, and for the entire genome length. The main limitation of this technology is the higher cost and the possibility of introducing errors during the reverse transcription and amplification stages. When approaching the regulators, it is important to be aware of the sensitivity of the method of choice.
A phenotypic analysis helps correlate mutations with resistance. By comparing mutants and wild type viruses upon treatment with the same concentrations of drugs, it is possible to quantify the reduced susceptibility of the mutants: dividing the IC50 of the mutant by the IC50 of the wild type viruses gives the fold resistance change (also known as a shift in susceptibility). The phenotypic analysis is particularly powerful when combined with reverse-engineering systems, where individual mutations or combinations can be introduced systematically into a laboratory genetic background (either a virus molecular clone or a replicon), and their impact on resistance can be individually assessed.
Cross-resistance analysis
Antivirals against the same target (for instance, products from the same drug class) might cause the development of mutations that lead to decreased susceptibility to the other antiviral products. When drug combinations target the same protein or even the same complex, assessing the onset of cross-resistance is crucial. This analysis should be carried out using multiple clinical isolates and multiple conditions.
The race is always on
The main purpose of preclinical virus resistance studies is to determine whether the tested compound has a low or high resistance threshold, and the impact of the most common and reproducible mutations in causing resistance, as well as the order they tend to occur. However, what happens in vivo or in a clinical setting might be completely different, and this is why monitoring resistance becomes even more relevant and informative during clinical studies.
The bottom line – if there is one in this continuous race between viruses and drug development – is that there will always be new challenges as a compound moves to clinical trials. However, the knowledge acquired during preclinical studies, as well as the assays and systems developed specifically for the virus of interest, will minimize the unknowns and thereby accelerating progression to the clinics (and reducing cost along the way).
