Vesicular Stomatitis Virus Chimeras Expressing the Oropouche Virus Glycoproteins Elicit Protective Immune Responses in Mice

Abstract

Oropouche virus (OROV) infection of humans is associated with a debilitating febrile illness that can progress to meningitis or encephalitis. First isolated from a forest worker in Trinidad and Tobago in 1955, the arbovirus OROV has since been detected throughout the Amazon basin with an estimated 500,000 human infections over 60 years. Like other members of the family Peribunyaviridae, the viral genome exists as 3 single-stranded negative-sense RNA segments. The medium-sized segment encodes a viral glycoprotein complex (GPC) that is proteolytically processed into two viral envelope proteins, Gn and Gc, responsible for attachment and membrane fusion. There are no therapeutics or vaccines to combat OROV infection, and we have little understanding of protective immunity to infection. Here, we generated a replication competent chimeric vesicular stomatitis virus (VSV), in which the endogenous glycoprotein was replaced by the GPC of OROV. Serum from mice immunized by intramuscular injection with VSV-OROV specifically neutralized wild-type OROV, and using peptide arrays we mapped multiple epitopes within an N-terminal variable region of Gc recognized by the immune sera. VSV-OROV lacking this variable region of Gc was also immunogenic in mice producing neutralizing sera that recognize additional regions of Gc. Challenge of both sets of immunized mice with wild-type OROV shows that the VSV-OROV chimeras reduce wild-type viral infection and suggest that antibodies that recognize the variable N terminus of Gc afford less protection than those that target more conserved regions of Gc. IMPORTANCE Oropouche virus (OROV), an orthobunyavirus found in Central and South America, is an emerging public health challenge that causes debilitating febrile illness. OROV is transmitted by arthropods, and increasing mobilization has the potential to significantly increase the spread of OROV globally. Despite this, no therapeutics or vaccines have been developed to combat infection. Using vesicular stomatitis (VSV) as a backbone, we developed a chimeric virus bearing the OROV glycoproteins (VSV-OROV) and tested its ability to elicit a neutralizing antibody response. Our results demonstrate that VSV-OROV produces a strong neutralizing antibody response that is at least partially targeted to the N-terminal region of Gc. Importantly, vaccination with VSV-OROV reduces viral loads in mice challenged with wild-type virus. These data provide novel evidence that targeting the OROV glycoproteins may be an effective vaccination strategy to combat OROV infection.

Keywords: Oropouche virus; arbovirus; bunyavirus; emerging infectious diseases; vaccines.

FIG 1

Generation and characterization of VSV-OROV. (A) Genomic organization of VSV-eGFP and VSV-OROV. Viral genomes are shown in the 3′ to 5′ orientation. The five viral genes, N (nucleocapsid), P (phosphoprotein), M (matrix), G (glycoprotein), and L (large polymerase), are shown. Enhanced green fluorescent protein (eGFP) is located in the first position and serves as a marker for infection. The VSV glycoprotein was replaced with the gene encoding the OROV M segment to generate VSV-OROV. Representative plaque assays are shown on the right as well as the endpoint titers in BSRT7 cells. (B) SDS-PAGE analysis of purified virions stained with Coomassie blue. Viral proteins are indicated on the right. (C) Electron micrographs of purified virions stained with 2% PTA. (D) Measurements of individual viral particles were carried out from micrographs like those shown in panel C. Each circle represents a single virion, and the line denotes the mean with the standard deviation (n = 35). Length and width differences between VSV and VSV-OROV particles were statistically significant; ****, P < 0.0001.

FIG 2

Generation of VSV-OROV chimeras and mutants. (A) Genomic organization of VSV-OROV, VSV-OROVΔ4, and VSV-OROVΔ8. Amino acids 484 to 698 were deleted from the N-terminal region of Gc to generate VSV-OROVΔ4, and amino acids 484 to 870 were deleted to generate VSV-OROVΔ8. Plaque assays of VSV-OROV (shown also in Fig. 1) and VSV-OROVΔ4 on BSRT7 cells are shown on the right. (B) VSV-OROVΔ8 isolates were passaged on BSRT7 cells, and the M segments from 5 isolates were sequenced by Sanger sequencing. Mutations found in the M segment are denoted on the right. (C) Growth curve of VSV, VSV-OROV, VSV-OROVΔ4, and VSV-OROVΔ8 Q1030*.

FIG 3

Inoculation of mice with VSV recombinants bearing OROV glycoproteins generates neutralizing antibodies. (A) Inoculation schedule of BALB/c mice. Animals (n = 5 per group) were immunized intramuscularly with VSV-eGFP, VSV-OROV, or VSV-OROVΔ4 and then boosted on day 28. Mice were sacrificed on day 35, and serum was collected. (B) Reciprocal dilutions of VSV or VSV-OROV serum that protects cells from 100 TCID₅₀ units of indicated viruses are shown (n = 3). (C) Reciprocal dilution of VSV or VSV-OROVΔ4 serum that protects cells from the viruses indicated (n = 3). No neutralization at the highest concentration of sera (1:100) was scored as 1. Statistical analysis was performed using an unpaired t test (*, P < 0.05; **, P < 0.01; ****, P < 0.0001). (D) Plaque reduction neutralization assays were performed to assess neutralization of wild-type OROV. Plaques were visually scored after incubation with serial dilutions of mouse serum. Each line (numbered 1 to 5) represents an individual mouse.

FIG 4

VSV-OROV and VSV-OROVΔ4 antibodies target Gn and Gc. (A) Schematic of the OROV M segment showing the two glycoproteins (Gn and Gc) as well as the nonstructural, NSm protein. Peptides identified in the peptide array with VSV-OROV are indicated in gray boxes, while peptides identified in the VSV-OROVΔ4 peptide array are indicated above the schematic in red lines and numbers. (B) Heat map of the VSV-OROV peptide array. Analysis of the peptide array was performed with five VSV-OROV mouse serum samples, one primary bleed sample, one VSV serum sample, and a sham control. Scale is shown in arbitrary units. (C) Mapping of the four identified peptides to the structure of the OROV Gc N-terminal head domain that was determined by Hellert et al. (16) (D) Peptide array from VSV-OROVΔ4 sera, like what is shown in panel B for VSV-OROV.

FIG 5

Prime-boost vaccination of mice with VSV recombinants and challenge with OROV. (A) Inoculation schedule of male, 6-week-old C57BL/6 mice. Mice were inoculated on day zero with 10⁶ FFU, boosted on day 28 with the same dose, and then challenged 1 week later with 10⁶ TCID₅₀ OROV (five mice per group). One week after challenge, mice were sacrificed. (B) Body weight (top) and temperature (bottom) were assessed each day following challenge with OROV. *, P < 0.001. (C) One day before challenge, VSV-OROV, VSV-OROVΔ4, and VSV sera were collected from mice and tested for neutralization of OROV. Each line (numbered 1 to 5) represents an individual mouse. (D) Viral loads were assessed by measuring S segment copies in blood and brain samples from mice following sacrifice. Brain samples were normalized to the internal control, HRPT1. *, P < 0.05.