Attenuated vesicular stomatitis viruses as vaccine vectors

Abstract

We showed previously that a single intranasal vaccination of mice with a recombinant vesicular stomatitis virus (VSV) expressing an influenza virus hemagglutinin (HA) protein provided complete protection from lethal challenge with influenza virus (A. Roberts, E. Kretzschmar, A. S. Perkins, J. Forman, R. Price, L. Buonocore, Y. Kawaoka, and J. K. Rose, J. Virol. 72:4704-4711, 1998). Because some pathogenesis was associated with the vector itself, in the present study we generated new VSV vectors expressing HA which are completely attenuated for pathogenesis in the mouse model. The first vector has a truncation of the cytoplasmic domain of the VSV G protein and expresses influenza virus HA (CT1-HA). This nonpathogenic vector provides complete protection from lethal influenza virus challenge after intranasal administration. A second vector with VSV G deleted and expressing HA (DeltaG-HA) is also protective and nonpathogenic and has the advantage of not inducing neutralizing antibodies to the vector itself.

FIG. 1

Recombinant VSV-influenza virus A/WSN constructs. A schematic representation of recombinant viruses indicating gene order is shown 3′ to 5′ on the negative-strand genomic RNA. Each intergenic region contains a transcriptional STOP/START signal recognized by the VSV polymerase (32). Restriction enzyme sequences used for constructing cDNAs of the recombinants are also shown.

FIG. 2

Immunoprecipitations of proteins from ³⁵S-labeled cell extracts. Cells infected with recombinant VSVs were metabolically labeled, lysed, and immunoprecipitated with mouse monoclonal antibodies specific for influenza virus A/WSN HA protein (A, lanes 2 to 8) or for VSV G protein (A, lane 1, and B). VSVΔG and VSVΔG-HA were from BHK-G cell extracts; all other samples were from BHK cell extracts. VSVrwt and VSV-HA recombinants were run on all gels for comparison of immunoprecipitated proteins. Mock, immunoprecipitations of uninfected BHK cell lysates.

FIG. 3

Sucrose-purified recombinant virions. Cells infected with recombinant viruses were metabolically labeled with ³⁵S translabel. Supernatants were collected, and viruses were purified by centrifugation through 20% sucrose. The volumes loaded were normalized for N-P protein content. Lane 1, 1% of total VSV-HA virus from a 3.5-cm-diameter dish of BHK cells; lane 2, 18% of total CT1-HA virus from a 3.5-cm-diameter dish of BHK cells, lane 3, 14% of total VSVΔG-HA virus from a 3.5-cm-diameter dish of BHK-G cells; lane 4, 13% of total VSVΔG virus from a 3.5-cm-diameter dish of BHK-G cells.

FIG. 4

Immunogold labeling of sucrose-purified recombinants. Electron micrographs of recombinant VSVrwt (A) and VSVΔG-HA (B) bound to carbon-coated grids, labeled with mouse monoclonal antibody to HA and secondary gold-conjugated antibody, and negatively stained.

FIG. 5

Attenuated recombinants provide protection from lethal influenza virus challenge. Average daily weights of mice inoculated with VSV recombinants and challenged with influenza virus A/WSN are shown. (A) Mice were inoculated intranasally with VSV-HA (n = 5) or CT1-HA (n = 5) at 5 × 10⁴ PFU/mouse on day 0. The mice were boosted with an equal amount of inoculum on day 21. The mice were challenged with a lethal dose of influenza virus A/WSN on day 35. (B) Conditions were the same as for panel A, except the mice were inoculated and boosted with VSVΔG (n = 4) or VSVΔG-HA (n = 5) at 5 × 10⁴ PFU/mouse. The error bars indicate ±0.5 × standard deviation.