A vaccine candidate for eastern equine encephalitis virus based on IRES-mediated attenuation
Introduction
Viruses in the genus Alphavirus, family Togaviridae, have positive strand RNA genomes and cause a spectrum of diseases that includes fever, rash, arthritis, meningitis and encephalitis. Most alphaviruses are mosquito-borne, and transmitted in nature among zoonotic vertebrate reservoir hosts, including birds, primates and rodents. Based on their cross reactivity, several antigenic complexes including the EEE, Venezuelan (VEE) and western equine encephalitis (WEE) complexes comprise the Alphavirus genus [1], [2]. EEEV is considered the most deadly of all the alphaviruses due to the high case fatality rates associated with infections, reaching as high as 90% in horses. In humans, the estimated case fatality rate approaches 80% and many survivors exhibit crippling sequelae such as mental retardation, convulsions, and paralysis that require life-long institutionalized care [3], [4].
Based on antigenic and genetic analyses, EEEV is classified into 4 subtypes: subtype 1 includes strains from North America (NA), whereas the remaining 3 are found in Central and South America (SA) [5], [6]. In general, EEEV strains from SA appear to be less virulent for humans than NA strains [7]. The former can occasionally cause disease and death in horses, but human infections are rarely recognized. In contrast, NA strains are genetically conserved, uniformly virulent and cause severe encephalitis in both humans and equids [8], [9], [10]. However, in mice, both NA and SA strains of EEEV are highly virulent and cause mortality rates as high as 70–90% following subcutaneous, intraperitoneal or intramuscular infection [11], [12]. In outbred laboratory mice, EEEV produces neurologic disease that resembles that following human and equine infections. Virus has been detected in the brain as early as day 1 post-infection (PI), and signs of disease are evident as early as days 3–4. Clinical signs of murine disease include ruffled hair, anorexia, vomiting, lethargy, posterior limb paralysis, convulsions, and coma. Histopathological studies reveal extensive involvement of the brain, including neuronal degeneration, cellular infiltration, and perivascular cuffing, similar to the pathological changes of the central nervous system that are described in naturally infected humans [11]. Immune protection against alphaviruses has been attributed mainly to the humoral response, with titers of neutralizing antibodies directly proportional to the level of protection against disease upon challenge [13], [14], [15], [16].
Despite over 65 years of research there is no licensed human vaccine or effective antiviral treatment available for human EEE, and control depends on mosquito abatement measures and avoidance of exposure to mosquito bites. Several live-attenuated candidate vaccines derived from the wild-type, virulent EEEV were assessed for their safety and efficacy in mice [17], [18] but have not been developed further. A formalin-inactivated vaccine prepared from a wild-type NA strain of EEEV [19] under Investigational New Drug (IND) status is currently available only for researchers and military personnel [20], and a similar inactivated vaccine is sold for equids and other domesticated animals [21], [22], [23], [24], [25], [26]. Recently, the U.S. Department of Agriculture licensed a three-component vaccine composed of inactivated VEE, EEE and WEE viruses, for use in equids [27], [28]. However, all of these inactivated vaccines suffer from poor immunogenicity and from the risk of residual live virus in the vaccine lot [29].
In addition to its importance as a natural pathogen of humans and domesticated animals, EEEV is a potent biological weapon [30], adding further urgency to the need for an effective vaccine. Therefore, we sought to develop an attenuated EEEV strain that would induce rapid, robust and long-lived immunity after a single vaccination. Earlier, Wang et al. developed two chimeric alphavirus strains (SIN/EEEV), both of which replicate efficiently in mammalian and mosquito cell cultures without any adaptation [31]. These vaccines were shown to be highly attenuated, immunogenic and efficacious against EEEV challenge in murine testing. Another chimeric alphavirus, which is derived from a cDNA clone encoding the western equine encephalitis virus (WEEV) nonstructural polyprotein and the EEEV structural polyprotein protects mice against EEEV but not against WEEV challenge [32]. Because the recombinant nature of these chimeras is believed to confer the attenuation phenotype, rather than point mutations that are subject to high rates of reversion, these chimeric viruses appear to be stably attenuated [33]. However, another safety concern for live, genetically engineered vaccines against arboviral diseases is that recombinant viruses might evolve in unforeseen and unexpected ways if they inadvertently underwent vector-borne circulation in the nature. The isolation of the VEEV vaccine strain, TC-83, from naturally infected mosquitoes collected in Louisiana during a 1971 Texas epidemic demonstrated the risk of transmission of attenuated alphaviruses [34], and experimental infections of the chimeric EEE vaccine strains demonstrated some residual mosquito infectivity [35].
To eliminate potential mosquito infectivity of live alphavirus vaccine strains, the findings of Finkelstein et al., that insect cells do not support efficient internal translation initiation from an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) [36], were exploited in another vaccine design [37]. The VEEV vaccine strain TC-83 was modified in cDNA clone form to replace the subgenomic promoter with EMCV IRES to drive expression of the structural protein genes from genomic RNA in mammalian cells. The resulting strain is incapable of infecting mosquitoes, and is also further attenuated for mice. Beginning with a wild-type backbone of chikungunya virus, this IRES approach produced a vaccine candidate that appears to have an optimal balance of immunogenicity and attenuation, combined with a lack of mosquito infectivity [38]. In addition to EMCV IRES, attenuation and elimination of mosquito infectivity was also dependent on inactivated subgenomic promoter as previously reported by Volkova et al., and Plante et al. Northern blot analysis performed by both these groups confirmed that insertion of several synonymous point mutations efficiently inactivated the subgenomic promoter [37], [38].
Considering these promising results, we utilized the EMCV IRES approach to generate an attenuated, mosquito-incompetent EEEV vaccine candidate. This recombinant virus was evaluated in mice to assess attenuation, immunogenicity and protection against EEEV challenge, and was also tested for its ability to infect mosquito cells and mosquitoes in vivo. The strong attenuation and mosquito-incompetent phenotypes we measured further support the promise of this approach for alphavirus vaccine development.
Section snippets
Cell lines
Baby hamster kidney (BHK-21) and Vero cells were obtained from the American Type Culture Collection (ATCC, Bethesda, MD) and grown at 37 °C in Eagle's minimal medium (MEM) with 5% fetal bovine serum (FBS) and 0.005% gentamycin sulphate. The mosquito cell line C6/36, also from the ATCC, was maintained in MEM at 32 °C with 5% FBS and 10% tryptose phosphate broth.
Virus
Wild-type (WT) North American EEEV strain FL93-939 was used for the development of the recombinant construct and also for all challenge
Recombinant virus
In the recombinant virus EEEV/IRES, the subgenomic promoter was inactivated by 16 synonymous point mutations, which prevented the reversion to an active SG RNA promoter. To promote synthesis of the EEEV structural proteins, the IRES sequence was cloned to replace the 5′ UTR in the subgenomic RNA. The infectivity of the recombinant viral EEEV/IRES RNA as determined by infectious center assay was 5 × 105 PFU/4 μg, which is comparable to that of other alphavirus cDNA clones [37], [41]. A small and
Discussion
Traditionally, live-attenuated RNA virus vaccines have been generated by serial passage through cell cultures, which results in attenuation that is dependent on a small numbers of point mutations [43], [44], [45], [46]. Because of the instability of the genome during RNA virus replication, these point mutations are likely to revert in vivo, or the virus may acquire a compensatory mutation(s) that can restore virulence. The live-attenuated VEE vaccine, TC-83 is a good example. This vaccine,
Acknowledgements
We thank Rui Mei Yun for excellent technical assistance and Jing Huang for rearing mosquitoes and assistance with mosquito work. This work was supported by a grant from the National Institute of Allergy and Infectious Disease (NIAID) through the Western Regional Center of Excellence for Biodefense and Emerging Infectious Disease Research, National Institutes of Health (NIH) grant U54 AIO57156.
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