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Display of Domain III from Dengue 2 Envelope Protein on HBsAg Virus-like Particles Vectored by Measles Virus

Description

Dengue virus infects millions of people every year. Yet there is still no vaccine available to prevent it. Here we use a neutralizing epitope determinant on the dengue envelope (E)

Dengue virus infects millions of people every year. Yet there is still no vaccine available to prevent it. Here we use a neutralizing epitope determinant on the dengue envelope (E) protein as an immunogen to be vectored by a measles virus (MV) vaccine. However the domain III (DIII) of the dengue 2 E protein is too small to be immunogenic by itself. In order for it to be displayed on a larger particle, it was inserted into the amino terminus of small hepatitis B surface antigen (HBsAg, S) coding sequence. To generate the recombinant MV vector and verify the efficiency of this concept, a reverse genetics system was used where the MV vectors express one or two additional transcription units to direct the assembly of hybrid HBsAg particles. Two types of recombinant measles virus were produced: pB(+)MVvac2(DIII-S,S)P and pB(+)MVvac2(DIII-S)N. Virus recovered from pB(+)MVvac2(DIII-S,S)P was viable. An ELISA assay was performed to demonstrate the expression and secretion of HBsAg. Supernatant from MVvac2(DIII-S,S)P infected cells confirmed that hybrid HBsAg-domain III particles with a density similar to traditional HBsAg particles were released. Characteristics of the subviral particle have been analyzed for the successful incorporation of domain III. The replication fitness of the recombinant MV was evaluated using multi-step growth kinetics and showed reduced replication fitness when compared to the parental strain MVvac2. This demonstrates that viral replication is hindered by the addition of the two inserts into MV genome. Further analysis of MVvac2(DIII-S)N is needed to justify immune response studies in a small animal model using both of the generated recombinant vectors.

Contributors

Created

Date Created
  • 2014-05

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Searching for an HIV vaccine: a heterologous prime-boost system using replicating vaccinia virus and plant-produced virus-like particles

Description

The HIV-1 pandemic continues to cause millions of new infections and AIDS-related deaths each year, and a majority of these occur in regions of the world with limited access to

The HIV-1 pandemic continues to cause millions of new infections and AIDS-related deaths each year, and a majority of these occur in regions of the world with limited access to antiretroviral therapy. Therefore, an HIV-1 vaccine is still desperately needed. The most successful HIV-1 clinical trial to date used a non-replicating canarypox viral vector and protein boosting, yet its modest efficacy left room for improvement. Efforts to derive novel vectors which can be both safe and immunogenic, have spawned a new era of live, viral vectors. One such vaccinia virus vector, NYVAC-KC, was specifically designed to replicate in humans and had several immune modulators deleted to improve immunogenicity and reduce pathogenicity. Two NYVAC-KC vectors were generated: one expressing the Gag capsid, and one with deconstructed-gp41 (dgp41), which contains an important neutralizing antibody target, the membrane proximal external region (MPER). These vectors were combined with HIV-1 Gag/dgp41 virus-like particles (VLPs) produced in the tobacco-relative Nicotiana benthamiana. Different plant expression vectors were compared in an effort to improve yield. A Geminivirus-based vector was shown to increase the amount of MPER present in VLPs, thus potentially enhancing immunogenicity. Furthermore, these VLPs were shown to interact with the innate immune system through Toll-like receptor (TLR) signaling, which activated antigen presenting cells to induce a Th2-biased response in a TLR-dependent manner. Furthermore, expression of Gag and dgp41 in NYVAC-KC vectors resulted in activation of antiviral signaling pathways reliant on TBK1/IRF3, which necessitated the use of higher doses in mice to match the immunogenicity of wild-type viral vectors. VLPs and NYVAC-KC vectors were tested in mice, ultimately showing that the best antibody and Gag-specific T cell responses were generated when both components were administered simultaneously. Thus, plant-produced VLPs and poxvirus vectors represent a highly immunogenic HIV-1 vaccine candidate that warrants further study.

Contributors

Agent

Created

Date Created
  • 2016