Using ΦX174 to Model Recovery of Attenuated Viral Vaccines
Additional Funding Sources
The project described was supported by a student grant from the UI Office of Undergraduate Research.
Abstract
Attenuated viral vaccines, like the influenza vaccine, are known to effectively reduce the threat of infectious diseases. However, some of these attenuated viruses are only a few mutations from regaining virulence. This study uses bacteriophage ΦX174, a virus that infects E. coli bacteria, to model the effects that various types and degrees of attenuation have on the recovery of phage back to a more virulent form. Data from this experiment can be used to inform the development of attenuated vaccines that are safer and more robust. For this study, a library of 127 ΦX174 attenuated phage variants was constructed using three types of whole-gene synonymous recoding (Random, Shuffle, and Worst) in three genes (F, G and H) combined in every possible configuration. Each individual phage construct was then evolved for five passages in liquid culture. During each passage, the phage population was expanded 10,000-fold (from 104 to 108). At this population size most one-step mutations would have arisen, and should they exist, those beneficial mutations should have swept through the population. For the constructs that were successfully evolved and increased fitness over five generations, sequencing revealed SNPs in both recoded and wildtype regions that alone recovered between a third and half of the fitness lost by attenuation. This was observed across multiple constructs. While sequencing and successful evolution of all 127 constructs is ongoing, our results show that while attenuation involves hundreds of synonymous mutations, a single mutation alone can reverse much of the intentional fitness loss accomplished by attenuation.
Using ΦX174 to Model Recovery of Attenuated Viral Vaccines
Attenuated viral vaccines, like the influenza vaccine, are known to effectively reduce the threat of infectious diseases. However, some of these attenuated viruses are only a few mutations from regaining virulence. This study uses bacteriophage ΦX174, a virus that infects E. coli bacteria, to model the effects that various types and degrees of attenuation have on the recovery of phage back to a more virulent form. Data from this experiment can be used to inform the development of attenuated vaccines that are safer and more robust. For this study, a library of 127 ΦX174 attenuated phage variants was constructed using three types of whole-gene synonymous recoding (Random, Shuffle, and Worst) in three genes (F, G and H) combined in every possible configuration. Each individual phage construct was then evolved for five passages in liquid culture. During each passage, the phage population was expanded 10,000-fold (from 104 to 108). At this population size most one-step mutations would have arisen, and should they exist, those beneficial mutations should have swept through the population. For the constructs that were successfully evolved and increased fitness over five generations, sequencing revealed SNPs in both recoded and wildtype regions that alone recovered between a third and half of the fitness lost by attenuation. This was observed across multiple constructs. While sequencing and successful evolution of all 127 constructs is ongoing, our results show that while attenuation involves hundreds of synonymous mutations, a single mutation alone can reverse much of the intentional fitness loss accomplished by attenuation.