Coronavirus-based vectors are a promising system to genetically deliver multiple heterologous genes to specific target cells. First, coronaviruses are positive-stranded RNA viruses replicating in the cytoplasm without a DNA intermediary, making insertion of viral sequences into the host cell genome unlikely. Second, coronaviruses have the largest RNA genome known so far and a cloning capacity of more than 6 kb is expected. Third, coronaviruses display a unique transcription process resulting in the synthesis of 6-8 subgenomic mRNAs, encoding mainly the structural genes which can be replaced by multiple genes-of-interest. Fourth, the receptor of human coronavirus HCoV 229E is expressed on dendritic cells. Fifth, the natural route of coronavirus infection is the mucosal route. The long term objective of our study is to generate a novel HIV vaccine vector that (i) targets and activates DC (ii) displays sufficient cloning capacity for insertion of several HIV antigens and immunostimulatory cytokines, (iii) encodes for antigenic determinants that induce broadly neutralizing antibody responses, and (iv) can be applied via mucosal surfaces.
The specific aim of this application is to provide the proof-of-principle that coronavirus vectors can induce protective antiviral humoral and cellular immune responses in vivo. To this end, we will generate mouse hepatitis virus (MHV) vectors carrying various antigens (fusion protein of the LCMV GP33 epitope KAVYNFATC and green fluorescent protein, vesicular stomatitis virus glycoprotein (VSV-G), and HIV-p24) and immunostimulatory cytokine GM-CSF. Mice will be immunized with recombinant MHV virus-like particles via different routes and the induction of antiviral CTL and Th cell responses and the production of neutralizing antibodies will be tested using well-established read-out systems. Since MHV infects mucosal tissues and is able to infect DC, recombinant MHV vectors in the context of a murine model can serve as a paradigm for the development and evaluation of coronavirus vaccine vectors in humans.
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