Analysis of Venezuelan Equine Encephalitis Replicon Particles Packaged in Different Coats

Background: The Venezuelan equine encephalitis (VEE) virus replicon system was used to produce virus-like replicon particles (VRP) packaged with a number of different VEE-derived glycoprotein (GP) coats. The GP coat is believed to be responsible for the cellular tropism noted for VRP and it is possible that different VEE GP coats may have different affinities for cells. We examined VRP packaged in four different VEE GP coats for their ability to infect cells in vitro and to induce both humoral and cellular immune responses in vivo. Methodology/Principal Findings: The VRP preparations were characterized to determine both infectious units (IU) and genome equivalents (GE) prior to in vivo analysis. VRP packaged with different VEE GP coats demonstrated widely varying GE/IU ratios based on Vero cell infectivity. BALB/c mice were immunized with the different VRP based on equal GE titers and the humoral and cellular responses to the expressed HIV gag gene measured. The magnitude of the immune responses measured in mice revealed small but significant differences between different GP coats when immunization was based on GE titers. Conclusions/Significance: We suggest that care should be taken when alternative coat proteins are used to package vector-based systems as the titers determined by cell culture infection may not represent accurate particle numbers and in turn may not accurately represent actual in vivo dose

Structural protein coding region and location of glycoprotein mutations in different VEE viruses

<p>Structural protein coding region and location of glycoprotein mutations in different VEE viruses</p

GAG-specific ELISPOT analysis post boost.

<p>Splenocytes were isolated from individual animals and GAG specific gamma interferon ELISPOT assays were performed to determine the number of antigen-specific cytokine-secreting T cells. Error bars represent 1 standard error.</p

GAG-specific ELISA analysis post boost.

<p>Sera was collected from individual animals 1 week after the booster vaccination and GAG-specific ELISA analysis was performed. Error bars represent 1 standard error.</p

Comparison of genome equivalents

a<p>Infectious unit (IU) titer determined on Vero cells. IU/ml titer represented.</p>b<p>Genome equivalent (GE) titer determined by quantitative reverse transcription PCR. GE/ml titer represented.</p

GAG-specific ELISPOT analysis post prime.

<p>Splenocytes were isolated from individual animals and GAG specific gamma interferon ELISPOT assays were performed to determine the number of antigen-specific cytokine-secreting T cells. Error bars represent 1 standard error.</p

VEE GP coat mouse immunogenicity study design

a<p>Genome equivalent (GE) titer determined by quantitative reverse transcription PCR.</p>b<p>Infectious unit (IU) titer determined on Vero cells.</p