Date Published: February 11, 2019
Publisher: Public Library of Science
Author(s): Derek W. Trobaugh, Chengqun Sun, Matthew D. Dunn, Douglas S. Reed, William B. Klimstra, Thomas E. Morrison.
Live attenuated vaccines (LAVs), if sufficiently safe, provide the most potent and durable anti-pathogen responses in vaccinees with single immunizations commonly yielding lifelong immunity. Historically, viral LAVs were derived by blind passage of virulent strains in cultured cells resulting in adaptation to culture and a loss of fitness and disease-causing potential in vivo. Mutations associated with these phenomena have been identified but rarely have specific attenuation mechanisms been ascribed, thereby limiting understanding of the attenuating characteristics of the LAV strain and applicability of the attenuation mechanism to other vaccines. Furthermore, the attenuated phenotype is often associated with single nucleotide changes in the viral genome, which can easily revert to the virulent sequence during replication in animals. Here, we have used a rational approach to attenuation of eastern equine encephalitis virus (EEEV), a mosquito-transmitted alphavirus that is among the most acutely human-virulent viruses endemic to North America and has potential for use as an aerosolized bioweapon. Currently, there is no licensed antiviral therapy or vaccine for this virus. Four virulence loci in the EEEV genome were identified and were mutated individually and in combination to abrogate virulence and to resist reversion. The resultant viruses were tested for virulence in mice to examine the degree of attenuation and efficacy was tested by subcutaneous or aerosol challenge with wild type EEEV. Importantly, all viruses containing three or more mutations were avirulent after intracerebral infection of mice, indicating a very high degree of attenuation. All vaccines protected from subcutaneous EEEV challenge while a single vaccine with three mutations provided reproducible, near-complete protection against aerosol challenge. These results suggest that informed mutation of virulence determinants is a productive strategy for production of LAVs even with highly virulent viruses such as EEEV. Furthermore, these results can be directly applied to mutation of analogous virulence loci to create LAVs from other viruses.
Vaccines against virus pathogens have been licensed in the United States since 1914  and most are inactivated or live-attenuated viruses. Inactivated vaccines are viruses that have been killed using either formaldehyde (polio virus, influenza virus, and hepatitis A virus) or β-propiolactone (influenza virus) rendering the virus unable to replicate after vaccination . Live-attenuated vaccines (LAVs) are live viruses that either have been mutated, most commonly by blind passage (e.g., ), or exhibit host incompatibility to reduce virulence after vaccination . Smallpox, measles, mumps and rubella virus (MMR), varicella virus (chicken pox), rotavirus, and yellow fever virus vaccines are LAVs that are currently FDA approved [5,6].
LAVs are an effective tool in combating medically important pathogens. However, LAVs can induce adverse events in some individuals limiting their use and distribution. Historically, LAVs have been generated by blind passaging in cell culture or animal models until attenuation was achieved. This serial passaging led to the accumulation of mutations in the virus genome that decreased virus virulence. While the mutations could be identified by sequencing, their specific mechanisms of attenuation were rarely known. This has remained true for very widely used LAVs such as the YFV 17D LAV  or the poliovirus LAV , which have been given to hundreds of millions of individuals. For example, with the exception of a substitution to positive charge in the DIII loop of the YFV E protein that confers enhanced interactions with heparan sulfate , the molecular attenuation mechanisms conferred by some or all of the 31 specific mutations in YFV 17D LAV have not been well characterized . In addition, attenuating mutations selected by blind passage of LAVs are not designed to resist reversion and often involve single nucleotide changes [3,10–12]. This can lead to rapid reversion to non-attenuated phenotypes [15,40].