Polymerase Mechanism-Based Method of Viral Attenuation
Vaccines remain the most effective way of preventing infection and spread of infectious diseases. These prophylactics have been used for centuries but still to this day only three main design strategies exist: (1) live attenuated virus (LAV) vaccines, (2) killed or inactivated virus vaccines, (3) and subunit vaccines of the three, the most efficacious vaccines remain LAVs. LAVs replicate in relevant tissues, elicit strong cellular and humoral responses, and often confer lifelong immunity. While this vaccine strategy has produced the majority of successful vaccines in use today, there are also important safety concerns to consider with this approach. In the past, the development of LAVs has been empirical. Blind passage of viruses in various cell types results in the accumulation of multiple attenuating mutations leaving the molecular mechanisms of attenuation unknown. Also, due to the high error rate of RNA viruses and selective pressures of the host environment, these LAVs, derived from such viruses, can potentially revert back to wild-type virulence. This not only puts the vaccinee at risk, but if shed can put those that are unvaccinated at risk as well. While these vaccines have been successful there still remains a need for a rational design strategy by which to create additional LAVs.
One approach for rational vaccine design involves increasing the fidelity of the viral RdRp. Increased fidelity decreases the viral mutational frequency thereby reducing the genetic variation the virus needs in order to evade the host imposed bottlenecks to infection. While polymerase mutants exist which decrease viral mutation frequency the mutations are not in conserved regions of the polymerase, which doesn’t lend itself toward using a common mutant approach toward developing a universal vaccine strategy for all RNA viruses. We have identified a conserved lysine residue in the active site of the PV RdRp that acts as a general acid during nucleotide incorporation. Mutation from a lysine to an arginine results in a high fidelity polymerase that replicates slowly thus creating an attenuated virus that is genetically stable and less likely to revert to a wild-type phenotype. This chapter provides detailed methods in which to identify the conserved lysine residue and evaluating fidelity and attenuation in cell culture (in vitro) and in the PV transgenic murine model (in vivo).
Key wordsRNA virus RNA-dependent RNA polymerase Polymerase fidelity Live-attenuated virus Vaccine Attenuation Poliovirus Sequence homology
- 2.Fischer WA, Chason KD, Brighton M et al (2014) Live attenuated influenza vaccine strains elicit a greater innate immune response than antigenically-matched seasonal influenza viruses during infection of human nasal epithelial cell cultures. Vaccine 32:1761–1767PubMedCentralCrossRefPubMedGoogle Scholar
- 7.Korboukh VK, Lee CA, Acevedo A et al (2014) RNA virus population diversity: an optimum for maximal fitness and virulence. J Biol Chem 289:29531–29544Google Scholar
- 9.Eigen M (1971) Selforganization of matter and the evolution of biological macromolecules. Die Naturwissenschaften 58:465–523Google Scholar
- 13.Coffey LL, Vignuzzi M (2010) Host alternation of chikungunya virus increases fitness while restricting population diversity and adaptability to novel selective pressures. J Virol 85:1025–1035Google Scholar
- 24.Crotty S, Hix L, Sigal LJ et al (2002) Poliovirus pathogenesis in a new poliovirus receptor transgenic mouse model: age-dependent paralysis and a mucosal route of infection. J Gen Virol 83:1707–1720Google Scholar