Novel Insect-Specific Eilat Virus-Based Chimeric Vaccine Candidates Provide Durable, Mono- and Multivalent, Single-Dose Protection against Lethal Alphavirus Challenge

Abstract

Most alphaviruses are mosquito borne and exhibit a broad host range, infecting many different vertebrates, including birds, rodents, equids, humans, and nonhuman primates. Recently, a host-restricted, mosquito-borne alphavirus, Eilat virus (EILV), was described with an inability to infect vertebrate cells based on defective attachment and/or entry, as well as a lack of genomic RNA replication. We investigated the utilization of EILV recombinant technology as a vaccine platform against eastern (EEEV) and Venezuelan equine encephalitis viruses (VEEV), two important pathogens of humans and domesticated animals. EILV chimeras containing structural proteins of EEEV or VEEV were engineered and successfully rescued in Aedes albopictus cells. Cryo-electron microscopy reconstructions at 8 and 11 Å of EILV/VEEV and EILV/EEEV, respectively, showed virion and glycoprotein spike structures similar to those of VEEV-TC83 and other alphaviruses. The chimeras were unable to replicate in vertebrate cell lines or in brains of newborn mice when injected intracranially. Histopathologic examinations of the brain tissues showed no evidence of pathological lesions and were indistinguishable from those of mock-infected animals. A single-dose immunization of either monovalent or multivalent EILV chimera(s) generated neutralizing antibody responses and protected animals against lethal challenge 70 days later. Lastly, a single dose of monovalent EILV chimeras generated protective responses as early as day 1 postvaccination and partial or complete protection by day 6. These data demonstrate the safety, immunogenicity, and efficacy of novel insect-specific EILV-based chimeras as potential EEEV and VEEV vaccines.IMPORTANCE Mostly in the last decade, insect-specific viruses have been discovered in several arbovirus families. However, most of these viruses are not well studied and largely have been ignored. We explored the use of the mosquito-specific alphavirus EILV as an alphavirus vaccine platform in well-established disease models for eastern (EEE) and Venezuelan equine encephalitis (VEE). EILV-based chimeras replicated to high titers in a mosquito cell line yet retained their host range restriction in vertebrates both in vitro and in vivo In addition, the chimeras generated immune responses that were higher than those of other human and/or equine vaccines. These findings indicate the feasibility of producing a safe, efficacious, mono- or multivalent vaccine against the encephalitic alphaviruses VEEV and EEEV. Lastly, these data demonstrate how host-restricted, insect-specific viruses can be engineered to develop vaccines against related pathogenic arboviruses that cause severe disease in humans and domesticated animals.

Keywords: Eilat virus; alphavirus; emerging infectious diseases; vaccines.

FIG 1

Schematic diagrams of EILV chimeras. EILV chimeras were rescued in C7/10 cells, and 48 h postelectroporation (hpe) supernatants were harvested and titrated on C7/10 cells.

FIG 2

Virion morphology of EILV chimeras determined by cryo-electron (cryo-EM) microscopy. Cryo-EM micrographs of EILV/EEEV (A) and EILV/VEEV (B). An 11-Å and 8-Å cryo-EM single-particle reconstruction of EILV/EEEV (B) and EILV/VEEV (D).

FIG 3

(A) Micrographs of EILV/VEEV were taken after 0 weeks (left) and 115 weeks (right) of storage at 4°C. (B) Micrographs of EILV/CHIKV were collected after 0 weeks (left) and 53 weeks (right) of storage at 4°C.

FIG 4

Electroporation of vertebrate and mosquito cell lines with EILV reporter chimeras. RNA was transcribed in vitro, each cell line was electroporated with ∼10 μg of RNA, and phase-contrast (left) and fluorescence (right) micrographs were taken at 1 and 4 days postelectroporation (dpe).

FIG 5

Neurovirulence of EILV chimeras in newborn mice. (A) Outline of the experimental design. (B and C) Percent survival (B) and virus replication (C) following intracranial inoculation of EILV chimeras, VEEV-TC83, and sucrose-purified C7/10 cell supernatants. Average virus titers are shown with each data point representing brain homogenate from 3 animals ± standard deviations (SD). *, P < 0.0001.

FIG 6

Histopathology of brain tissues. Micrographs of hematoxylin- and eosin-stained sections of brain tissues following intracranial inoculation of EILV chimeras, VEEV-TC83, and sucrose-purified C7/10 cell supernatants are shown. Three animals were sacrificed at the time points indicated, and tissues were stained to determine necrotic and inflammatory lesions. Necrotic and inflammatory lesions in VEEV-TC83 were magnified (40×) and are shown.

FIG 7

Immunogenicity and efficacy of monovalent EILV/EEEV chimera in CD-1 mice. (A) Outline of the experimental design. (B) Neutralizing antibody response measured by PRNT₈₀ titers following EILV/EEEV vaccination. Percent weight loss (C) and survival (D) following lethal EEEV-NA challenge via the subcutaneous route (SC). Average PRNT₈₀ titers and weight losses ± standard deviations (SD) (error bars) are shown. *, P ≤ 0.01; **, P < 0.0001.

FIG 8

Immunogenicity and efficacy of monovalent EILV/VEEV chimera in CD-1 mice. (A) Outline of the experimental design. (B) Neutralizing antibody response measured by PRNT₈₀ titers following EILV/VEEV vaccination. (B and C) Percent weight loss (C) and survival (D) following lethal VEEV-IC challenge via the subcutaneous route (SC). Average PRNT₈₀ titers and weight losses ± SD (error bars) are shown. *, P ≤ 0.04; **, P < 0.0001.

FIG 9

Immunogenicity and efficacy of blended multivalent EILV chimeras in CD-1 mice. (A) Outline of the experimental design. (B) Neutralizing antibody response measured by PRNT₈₀ titers following EILV/EEEV, EILV/VEEV, and EILV/CHIKV vaccination. (C and D) Percent survival and weight loss following lethal EEEV-NA (C) and VEEV-IC (D) challenge via the subcutaneous route (SC). Average PRNT₈₀ titers and weight losses ± SD (error bars) are shown. *, P ≤ 0.014.

FIG 10

Kinetics of protective immunity induced by monovalent EILV/EEEV chimera in CD-1 mice. (A) Outline of the experimental design. (B to D) Percent survival and weight loss following lethal EEEV-NA challenge via the subcutaneous route (SC) at 6 (B), 4 (C), and 1 (D) day postvaccination. Average weight losses ± SD (error bars) are shown. *, P ≤ 0.04.

FIG 11

Kinetics of protective immunity induced by monovalent EILV/VEEV chimera in CD-1 mice. (A) Outline of the experimental design. (B to D) Percent survival and weight loss following lethal VEEV-IC challenge via the subcutaneous route (SC) at 6 (B), 4 (C), and 1 (D) day postvaccination. Average weight losses ± SD (error bars) are shown. *, P < 0.0006.