Recombinant Isfahan Virus and Vesicular Stomatitis Virus Vaccine Vectors Provide Durable, Multivalent, Single-Dose Protection against Lethal Alphavirus Challenge

Abstract

The demonstrated clinical efficacy of a recombinant vesicular stomatitis virus (rVSV) vaccine vector has stimulated the investigation of additional serologically distinct Vesiculovirus vectors as therapeutic and/or prophylactic vaccine vectors to combat emerging viral diseases. Among these viral threats are the encephalitic alphaviruses Venezuelan equine encephalitis virus (VEEV) and Eastern equine encephalitis virus (EEEV), which have demonstrated potential for natural disease outbreaks, yet no licensed vaccines are available in the event of an epidemic. Here we report the rescue of recombinant Isfahan virus (rISFV) from genomic cDNA as a potential new vaccine vector platform. The rISFV genome was modified to attenuate virulence and express the VEEV and EEEV E2/E1 surface glycoproteins as vaccine antigens. A single dose of the rISFV vaccine vectors elicited neutralizing antibody responses and protected mice from lethal VEEV and EEEV challenges at 1 month postvaccination as well as lethal VEEV challenge at 8 months postvaccination. A mixture of rISFV vectors expressing the VEEV and EEEV E2/E1 glycoproteins also provided durable, single-dose protection from lethal VEEV and EEEV challenges, demonstrating the potential for a multivalent vaccine formulation. These findings were paralleled in studies with an attenuated form of rVSV expressing the VEEV E2/E1 glycoproteins. Both the rVSV and rISFV vectors were attenuated by using an approach that has demonstrated safety in human trials of an rVSV/HIV-1 vaccine. Vaccines based on either of these vaccine vector platforms may present a safe and effective approach to prevent alphavirus-induced disease in humans.IMPORTANCE This work introduces rISFV as a novel vaccine vector platform that is serologically distinct and phylogenetically distant from VSV. The rISFV vector has been attenuated by an approach used for an rVSV vector that has demonstrated safety in clinical studies. The vaccine potential of the rISFV vector was investigated in a well-established alphavirus disease model. The findings indicate the feasibility of producing a safe, efficacious, multivalent vaccine against the encephalitic alphaviruses VEEV and EEEV, both of which can cause fatal disease. This work also demonstrates the efficacy of an attenuated rVSV vector that has already demonstrated safety and immunogenicity in multiple HIV-1 phase I clinical studies. The absence of serological cross-reactivity between rVSV and rISFV and their phylogenetic divergence within the Vesiculovirus genus indicate potential for two stand-alone vaccine vector platforms that could be used to target multiple bacterial and/or viral agents in successive immunization campaigns or as heterologous prime-boost agents.

Keywords: Eastern equine encephalitis virus; Isfahan virus; Venezuelan equine encephalitis virus; vesicular stomatitis virus.

FIG 1

Midpoint-rooted maximum likelihood tree based on nucleotide sequences of the N, G, and L genes. Bootstrap values of >75% are shown at internal nodes. The bar represents nucleotide substitutions per site.

FIG 2

(A) Schematic diagram of the strategy utilized to generate the rISFV genomic cDNA clone. The full-length viral genome was cloned in 5 fragments flanked by unique restriction sites. The 3′ leader and 5′ trailer were flanked by the T7 RNA polymerase promoter and terminator, respectively. The hepatitis delta virus ribozyme sequence was used to generate a precise 5′ trailer nucleotide sequence. (B) Outline of a helper-virus-free method of rescuing infectious rISFV. The T7 RNA polymerase and full-length and support rISFV plasmids were electroporated into Vero cells, and infectious virus was recovered at 5 to 10 days postelectroporation.

FIG 3

Schematic diagrams of the genetic organization and nomenclature of rISFV and rVSIV vectors. The N gene of rISFV-VEEV, rISFV-EEEV, and rVSIV-VEEV vectors was relocated to the fourth position of the genome. The rVSIV-VEEV vector also has a 28-amino-acid truncation in the cytoplasmic tail of the G protein. All foreign gene expression cassettes were added at genome position 5.

FIG 4

In vitro characterization of rISFV vectors. (A) Western blot analysis of rISFV and rVSIV vectors expressing VEEV or EEEV E3-E1 proteins in Vero cells. Replicate Vero cell monolayers in six-well plates were infected at an MOI of 5 PFU/cell, and cell lysates were collected at 24 hpi. The E3-E1 proteins were detected with mouse polyclonal antisera against VEEV and EEEV. (B and C) Plaque phenotype (B) and replication kinetics (C) of rISFV and rVSIV vectors in Vero cells. The plaque phenotype was assessed in Vero cell monolayers infected with wt ISFV, wt VSIV, rISFV, and rVSIV vectors, and at 2 to 3 days postinfection, the cells were fixed and stained with crystal violet. Assays of replication kinetics (C) of all viruses were performed at an MOI of 10 PFU/cell in triplicate. Average titers ± standard deviations (error bars) are shown. *, P values of <0.02.

FIG 5

Efficacy of the rISFV-VEEV vector in CD-1 mice. (A) Outline of the study design. (B and C) Percent weight loss (B) and survival (C) following lethal VEEV-ID challenge via the s.c. route. Average weight loss ± standard deviations (error bars) are shown. dpi, days postinfection.

FIG 6

Immunogenicity and efficacy of the rISFV-EEEV vector in CD-1 mice. (A and B) Outline of the study design (A) and neutralizing antibody response measured by a PRNT₈₀ assay following rISFV-EEEV vaccination (B). (C and D) Percent weight loss (C) and survival (D) following lethal EEEV-NA challenge via the s.c. route. Average PRNT₈₀ values and weight loss ± standard deviations (error bars) are shown.

FIG 7

Immunogenicity and efficacy of blended rISFV-VEEV/rISFV-EEEV vectors. (A) Outline of the study design in CD-1 mice. (B) Neutralizing antibody response measured by a PRNT₈₀ assay following vaccination. (C) Survival following lethal VEEV-ID or EEEV-NA challenge via the s.c. or i.p. route, respectively. Average PRNT₈₀ values ± standard deviations (error bars) are shown.

FIG 8

Dose titration and duration of efficacy of the rISFV-VEEV vector. (A) Outline of the study design in CD-1 mice. Animals were vaccinated with 10⁸ or 10⁷ PFU and challenged with lethal VEEV-ID at 35 and 245 days postinfection. (B) Neutralizing antibody response measured by a PRNT₈₀ assay following vaccination. (C) Survival following lethal VEEV-ID challenge via the s.c. route. Average PRNT₈₀ values ± standard deviations (error bars) are shown.

FIG 9

Dose titration and duration of efficacy of the rVSIV-VEEV vector. (A) Outline of the study design in CD-1 mice. Animals were vaccinated with 10⁸ or 10⁷ PFU and challenged with lethal VEEV-ID at 35 and 245 days postinfection. (B) Neutralizing antibody response measured by a PRNT₈₀ assay following vaccination. (C) Survival following lethal VEEV-ID challenge via the s.c. route. Average PRNT₈₀ values ± standard deviations (error bars) are shown.