Abstract
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.
Highlights
Vaccines against virus pathogens have been licensed in the United States since 1914 [1] and most are inactivated or live-attenuated viruses
Liveattenuated vaccines (LAVs) have been created by blind passaging of the virus leading to attenuating mutations in the viral genome with no known mechanism of action
We created a series of LAVs by combining mutations of four alphavirus virulence loci that have known functions
Summary
Vaccines against virus pathogens have been licensed in the United States since 1914 [1] and most are inactivated or live-attenuated viruses. Liveattenuated vaccines (LAVs) are live viruses that either have been mutated, most commonly by blind passage (e.g., [3]), or exhibit host incompatibility to reduce virulence after vaccination [4]. One LAV in particular, the yellow fever virus (YFV) 17D vaccine, induces a neutralizing antibody response in >95% of vaccinees that can persist for >35 years [8]. While effective in attenuating virulent viruses, serial passaging introduces mutations in the virus genome that have unknown mechanisms of action and can exhibit minimal genetic differences compared to virulent parental strains [5]. The LAV poliovirus vaccine strain contains 10 essential attenuating mutations [14] and reversion of these mutations yielding virulent viruses can occur rapidly in vaccinees [15] putting unimmunized populations at risk. LAVs are often contraindicated in young or immunocompromised populations because of safety concerns [16]
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