Rational design of a live-attenuated eastern equine encephalitis virus vaccine through informed mutation of virulence determinants

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. Author summary Live-attenuated vaccines (LAVs) mimic a natural virus infection and elicit high levels of neutralizing antibodies that can persist for long times. Historically, 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 have used an informed approach to create a LAV for eastern equine encephalitis virus (EEEV). EEEV is one of the most highly virulent mosquito-borne viruses in the United States, and there is currently no approved vaccine or antiviral therapeutic. Here, we created a series of LAVs by combining mutations of four alphavirus virulence loci that have known functions. We demonstrate that viruses containing at last three mutations are highly attenuated after both a subcutaneous and intracerebral infection of mice and provide protective immunity against both a subcutaneous and aerosol challenge. We have also identified a key mutation, elimination of the miR-142-3p microRNA biding sites in the EEEV 3' untranslated region, as critical for myeloid cell replication and essential for eliciting optimal cytokine responses, T cell responses, and protection from challenge. In summary, our results provide a rationale for an informed approach to the generation of LAVs against arboviruses.